Fermentation-membrane separation coupling production method of high-purity lysine

Abstract

The invention discloses a fermentation-membrane separation coupling production method of high-purity lysine, and belongs to the technical field of amino acids, the method comprises the following steps: fermentation: inoculating a corynebacterium glutamicum seed culture solution in a lysine fermentation culture medium, carrying out fermentation culture at 32-34 DEG C for 28-30 hours, and when the fermentation culture is carried out for 17-19 hours, carrying out membrane separation on the fermentation culture medium to obtain the high-purity lysine. N-(4-aminobutyl) acetamide is added; performing membrane separation: performing cross-flow filtration on the fermentation liquor by adopting a surface modified membrane, controlling the inlet pressure to be 0.4-0.5 MPa, the outlet pressure to be 0.2-0.3 MPa and the temperature to be 30-35 DEG C, and obtaining a lysine crude product after the membrane separation is finished; post-treatment: carrying out ion exchange and crystallization on the lysine crude product to obtain a high-purity lysine product; the release of viscous pollutants is reduced from a fermentation source, the pollution resistance of the membrane is improved, the flux recovery rate of the membrane is improved, and efficient coupling of fermentation and separation is realized.

Description

Technical Field

[0001] This invention belongs to the technical field of amino acids, specifically relating to a fermentation-membrane separation coupled production method for high-purity lysine. Background Technology

[0002] Lysine, an essential amino acid for humans and animals, has wide applications in feed, food, and pharmaceuticals. Currently, lysine is mainly produced industrially through microbial fermentation, with Corynebacterium glutamicum being a commonly used strain. However, traditional lysine fermentation processes still face many challenges.

[0003] During fermentation, as the process progresses, the metabolic stress on the cells increases, which can easily lead to cell membrane damage or even rupture, releasing large amounts of intracellular substances such as nucleic acids and proteins. These substances not only inhibit further acid production by the cells, making it difficult to improve the sugar-acid conversion rate, but also significantly increase the viscosity and complexity of the fermentation broth, posing significant challenges to subsequent separation and purification.

[0004] In the separation process, membrane separation technology, especially ceramic membrane ultrafiltration, is widely used for cell separation in fermentation broths due to its advantages such as high temperature resistance, chemical corrosion resistance, and high mechanical strength. However, fermentation broths have complex compositions, containing various contaminants such as cell fragments, proteins, polysaccharides, and pigments, which are easily adsorbed and deposited on the membrane pores and surface during membrane filtration, causing severe membrane fouling. Membrane fouling leads to a sharp decline in membrane flux, requiring frequent shutdowns for cleaning. This not only significantly shortens the membrane's lifespan and increases production costs but also severely restricts production efficiency.

[0005] To mitigate membrane fouling, surface modification of separation membranes to enhance their hydrophilicity and antifouling capabilities is a common solution in existing technologies. Grafting hydrophilic polymers, such as polyethylene glycol brushes, is a mainstream method. However, such single-structure modified layers often fail to provide satisfactory protection against the complex composition of lysine fermentation broth. The contaminants in the fermentation broth exhibit a wide range of sizes, from large-molecule bacterial fragments to small-molecule pigments, making it difficult for a single polymer brush structure to effectively repel a large number of contaminants.

[0006] Therefore, developing a coupled production technology that can actively resist organic pollution and enhance the fermentation process is of great significance for achieving efficient and low-cost production of high-purity lysine. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a fermentation-membrane separation coupled production method for high-purity lysine, which reduces the release of sticky contaminants at the fermentation source, improves the membrane's antifouling resistance, increases the membrane's flux recovery rate, and achieves efficient coupling of fermentation and separation.

[0008] To solve the above technical problems, the technical solution adopted by the present invention is as follows: (1) Fermentation: In lysine fermentation medium, inoculate with Corynebacterium glutamicum seed culture medium and ferment at 32-34℃ for 28-30h. When fermentation is 17-19h, add N-(4-aminobutyl)acetamide. The preparation of the Corynebacterium glutamicum seed culture medium includes activation culture and seed culture; The activation culture method is as follows: Corynebacterium glutamicum is inoculated into activation culture medium and cultured at 30-32℃ for 23-25 ​​hours. The Corynebacterium glutamicum was purchased from the market. The activated culture medium consists of 9-11 g / L glucose, 9-11 g / L peptone, 8-10 g / L ammonium sulfate, 2.0-2.4 g / L KH2PO4, 2-2.5 g / L sodium chloride, and has a pH of 6.8-7.2. The seed culture method is as follows: the activated bacterial solution is inoculated into the seed culture medium at an inoculation amount of 4.5-5.5% by volume for seed culture, the culture temperature is 28-30℃, the culture time is 19-21h, and the seed culture solution is obtained after the culture is completed. The seed culture medium consists of 75-85 g / L glucose, 8-10 g / L ammonium sulfate, 15-20 g / L corn steep liquor powder, 1.5-2.5 g / L KH2PO4, 0.9-1.1 g / L MgSO4·7H2O, and a pH of 6.8-7.2. The amount of N-(4-aminobutyl)acetamide added is 0.2-0.3 g / L; The fermentation medium consists of 55-65 g / L glucose, 10-15 g / L ammonium sulfate, 34-36 g / L corn steep liquor powder, 2.8-3.2 g / L KH2PO4, 1.4-1.6 g / L MgSO4·7H2O, and a pH of 6.8-7.2. In the fermentation step, the amount of seed culture medium added is 5.5-6.5% of the volume of the fermentation medium.

[0009] (2) Membrane separation: The fermentation broth is filtered by cross-flow using a surface-modified membrane with an inlet pressure of 0.4-0.5 MPa and an outlet pressure of 0.2-0.3 MPa. The temperature is controlled at 30-35℃. Crude lysine is obtained after membrane separation. The bacterial cells retained after membrane separation are washed, activated, and concentrated before being used for inoculation in the next batch of fermentation.

[0010] (3) Post-processing: The crude lysine is subjected to ion exchange and crystallization to obtain a high-purity lysine product; In the ion exchange step, the resin type is cation exchange resin, and the pH of crude lysine is adjusted to 3.5-4.5 with 10-15% hydrochloric acid solution. The volume percentage of resin to fermentation broth is 1:2.8-3.2. The crystallization method is as follows: the eluent obtained by ion exchange is concentrated to 550-650 g / L at 60-70℃, and then the concentrated solution is cooled to 10-15℃ at 1-1.5℃ / h to obtain wet crystals. The wet crystals are dried to obtain the lysine product.

[0011] The method for preparing the surface-modified film includes the following steps: film surface activation, silanization, and polymer grafting; Membrane surface activation: The ceramic membrane was treated with H2O2 solution, and after cleaning and drying, an activated membrane was obtained. The ceramic membrane has a ZrO2 composition and a pore size of 40-60 nm. The concentration of the H2O2 solution is 3-4 wt%; The temperature for treating the ceramic membrane with the H2O2 solution is 38-42℃, and the treatment time is 30-35 min. The drying method is to dry at 70-75℃ for 3-3.5 hours.

[0012] Silanization: The activated membrane is immersed in a solution of γ-glycidyl oxypropyltrimethoxysilane and reacted at 58-62℃ for 4.8-5.2 hours. Then it is ultrasonically cleaned with ethanol 2-3 times and dried to obtain the silanized membrane. The solvent for the γ-glycidyl etheroxypropyltrimethoxysilane solution is an ethanol-water mixture with a concentration of 1.5-2 wt%. The mass ratio of ethanol to water in the ethanol-water mixture is 93-96:4-7; The ultrasonic frequency for the ethanol ultrasonic cleaning is 40-60kHz.

[0013] Polymer grafting: The silanized film is immersed in a poly(2-hydroxypropyl)methacrylamide solution, boron trifluoride diethyl ether is added, and the reaction is carried out at 68-72℃ for 3.8-4.2 hours; it is then ultrasonically cleaned in water at 40-45℃ 3-4 times, and dried at 45-50℃ for 11-13 hours to obtain a surface-modified film. The preparation of the poly(2-hydroxypropyl)methacrylamide solution involves dissolving low molecular weight poly(2-hydroxypropyl)methacrylamide and high molecular weight poly(2-hydroxypropyl)methacrylamide in water, wherein the molar ratio of low molecular weight poly(2-hydroxypropyl)methacrylamide to high molecular weight poly(2-hydroxypropyl)methacrylamide is 87-89:11-13, and the total concentration is 0.8-1 mol / L; The low molecular weight poly(N-(2-hydroxypropyl)methacrylamide) has a molecular weight of 800-1200; The high molecular weight poly(N-(2-hydroxypropyl)methacrylamide) has a molecular weight of 25,000-35,000; The mass ratio of boron trifluoride ether to poly(N-(2-hydroxypropyl)methacrylamide) solution is 0.4-0.6:100.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In this invention, N-(4-aminobutyl)acetamide is added during the middle stage of fermentation. This additive can stabilize the cell membrane structure and reduce the damage to the cells caused by the high metabolic load in the later stage of fermentation, thereby effectively reducing the cell breakage rate. This not only maintains the continuous acid production capacity of the cells and improves the sugar-acid conversion rate, but more importantly, it significantly reduces the release of viscous pollutants such as nucleic acids and intracellular proteins from the source, reduces the viscosity of the fermentation broth at the end of fermentation, creates more favorable conditions for downstream membrane separation, and achieves efficient coupling of fermentation and separation.

[0015] (2) This invention achieves excellent antifouling effect by constructing a synergistic graft layer on the surface of a ceramic membrane composed of a specific ratio of low molecular weight (800-1200) and high molecular weight (25000-35000) poly-N-(2-hydroxypropyl)methacrylamide. This dual molecular weight synergistic structure can form a dual barrier on the membrane surface that combines density and flexibility. The low molecular weight polymer helps to form a dense basic hydration layer, effectively blocking the adsorption of small molecule organic matter; while the high molecular weight polymer interspersed therein, through its longer chain segments and stronger steric hindrance effect, under high shear force, washes away large-volume pollutants such as bacteria, bacterial fragments, and colloids by oscillation.

[0016] (3) The purity of the lysine product prepared by the method of the present invention is 98.5-98.8%; the sugar-acid conversion rate of lysine fermentation is 65.5-66.0%; and the viscosity of the fermentation broth at the end of fermentation is 5.0-5.3 mPa·s. When the surface-modified membrane prepared by the method of the present invention is used in the fermentation-membrane separation coupled production method of the present invention, after continuous operation for 4 hours, the flux decline rate is 13.8-14.1%; after continuous operation for 4 hours, the surface-modified membrane is rinsed with 1.0wt% NaOH solution and 1.0wt% HCl solution for 30 min each, and then rinsed with water until the pH of the rinsing solution is 7.0, and the pure water flux recovery rate of the membrane is 96.3-96.9%. Detailed Implementation

[0017] To provide a clearer understanding of the technical features, objectives, and effects of the present invention, specific embodiments of the present invention are now described.

[0018] Example 1 Preparation of surface-modified films: (1) Activation of membrane surface: The ceramic membrane was treated with a 3.5wt% H2O2 solution at 40℃ for 32 min, washed with water and dried at 72℃ for 3.2 h to obtain the activated membrane; The ceramic membrane is composed of ZrO2 and has a pore size of 50 nm.

[0019] (2) Silanization: The activated membrane was immersed in a 1.8wt% γ-glycidyl oxypropyltrimethoxysilane solution and reacted at 60°C for 5 hours. Then it was ultrasonically cleaned three times with ethanol at a frequency of 50kHz and dried to obtain the silanized membrane. The solvent for the γ-glycidyl etheroxypropyltrimethoxysilane solution is an ethanol-water mixture with a mass ratio of ethanol to water of 95:5.

[0020] (3) Polymer grafting: The silanized film was immersed in poly(2-hydroxypropyl)methacrylamide solution, boron trifluoride ether was added, and the reaction was carried out at 70°C for 4 hours; it was ultrasonically cleaned in water at 42°C 3 times, and dried at 48°C for 12 hours to obtain the surface modified film. The poly(2-hydroxypropyl)methacrylamide solution is prepared by dissolving low molecular weight poly(2-hydroxypropyl)methacrylamide (1000) and high molecular weight poly(2-hydroxypropyl)methacrylamide (30000) in water, wherein the molar fraction of low molecular weight poly(2-hydroxypropyl)methacrylamide is 88%, the molar fraction of high molecular weight poly(2-hydroxypropyl)methacrylamide is 12%, and the total concentration is 0.9 mol / L. The mass ratio of boron trifluoride ether to poly(N-(2-hydroxypropyl)methacrylamide) solution is 0.5:100.

[0021] Comparative Example 1 Comparative Example 1 uses the surface-modified film preparation method described in Example 1, except that in the polymer grafting step, the preparation of the poly-N-(2-hydroxypropyl)methacrylamide solution uses only low molecular weight poly-N-(2-hydroxypropyl)methacrylamide with a molecular weight of 500, omitting high molecular weight poly-N-(2-hydroxypropyl)methacrylamide, while the other steps are the same.

[0022] Example 2 A fermentation-membrane separation coupled production method for high-purity lysine includes the following steps: (1) Fermentation Activation culture: Commercially purchased Corynebacterium glutamicum was inoculated into activation culture medium and cultured at 30℃ for 25 h. The activation culture medium consisted of: glucose 9 g / L, peptone 9 g / L, ammonium sulfate 8 g / L, KH2PO4 2.0 g / L, sodium chloride 2 g / L, pH 7.2.

[0023] Seed culture: The activated bacterial solution was inoculated into the seed culture medium at an inoculation rate of 4.5% (v / v) and cultured at 28℃ for 21 h. The seed culture medium consisted of: glucose 75 g / L, ammonium sulfate 8 g / L, corn steep liquor powder 15 g / L, KH2PO4 1.5 g / L, MgSO4·7H2O 0.9 g / L, pH 7.2.

[0024] Fermentation culture: The seed culture was inoculated into the fermentation medium at an inoculation rate of 5.5% (v / v) and fermented at 32°C for 30 h. At 19 h of fermentation, N-(4-aminobutyl)acetamide was added at a concentration of 0.2 g / L. The fermentation medium consisted of: glucose 55 g / L, ammonium sulfate 10 g / L, corn steep liquor powder 34 g / L, KH₂PO₄ 2.8 g / L, MgSO₄·7H₂O 1.4 g / L, and pH 7.2.

[0025] (2) Membrane separation After fermentation, the fermentation broth was directly introduced into a membrane separation system composed of surface-modified membranes prepared in Example 1. The inlet pressure was controlled at 0.4 MPa, the outlet pressure at 0.2 MPa, and the system temperature at 30°C. Cross-flow filtration was performed to obtain a clear crude lysine solution. The cells retained by membrane separation were washed, activated, and concentrated before being used for the next batch of fermentation inoculation.

[0026] (3) Post-processing Ion exchange: The crude lysine was adjusted to pH 4.5 with 10% hydrochloric acid solution, and then adsorbed and eluted through a cation exchange resin. The volume percentage of resin to fermentation broth was 1:3.2.

[0027] Crystallization and drying: The eluent obtained from ion exchange was concentrated to 550 g / L under vacuum at 60°C. The concentrate was then cooled to 15°C at a programmed rate of 1°C / h to crystallize. The crystals were then centrifuged to obtain wet crystals. After drying, high-purity lysine was obtained.

[0028] The lysine product prepared in this example has a purity of 98.5%.

[0029] Fermentation performance: Using glucose in the fermentation medium as the substrate, the sugar-acid conversion rate was calculated by detecting the lysine concentration in the fermentation broth at the fermentation endpoint. In this example, the sugar-acid conversion rate of lysine fermentation was 65.7%; the viscosity of the fermentation broth at the fermentation endpoint was 5.2 mPa·s.

[0030] Membrane separation performance: Under the operating conditions of the membrane separation step in this embodiment, the flux decline rate of the surface-modified membrane was 13.8% after 4 hours of continuous operation; After 4 hours of continuous operation, the surface-modified membrane was rinsed for 30 minutes each with 1.0 wt% NaOH solution and 1.0 wt% HCl solution, and then rinsed with water until the pH of the rinsing solution was 7.0. The pure water flux recovery rate of the membrane was 96.7%.

[0031] Example 3 A fermentation-membrane separation coupled production method for high-purity lysine includes the following steps: (1) Fermentation Activation culture: Commercially available Corynebacterium glutamicum was inoculated into activation medium and cultured at 31°C for 24 hours. The activation medium consisted of: 10 g / L glucose, 10 g / L peptone, 9 g / L ammonium sulfate, 2.2 g / L KH₂PO₄, 2.2 g / L sodium chloride, and pH 7.0.

[0032] Seed culture: The activated bacterial solution was inoculated into the seed culture medium at an inoculation rate of 5.0% (v / v) and cultured at 29℃ for 20 h. The seed culture medium consisted of: 80 g / L glucose, 9 g / L ammonium sulfate, 17 g / L corn steep liquor powder, 2.0 g / L KH2PO4, 1.0 g / L MgSO4·7H2O, and pH 7.0.

[0033] Fermentation culture: The seed culture was inoculated into the fermentation medium at an inoculation rate of 6.0% (v / v) and fermented at 33°C for 29 h. At 18 h of fermentation, N-(4-aminobutyl)acetamide was added at a concentration of 0.25 g / L. The fermentation medium consisted of: glucose 60 g / L, ammonium sulfate 12 g / L, corn steep liquor powder 35 g / L, KH₂PO₄ 3.0 g / L, MgSO₄·7H₂O 1.5 g / L, pH 7.0.

[0034] (2) Membrane separation After fermentation, the fermentation broth was directly introduced into a membrane separation system composed of the surface-modified membrane prepared in Example 1. The inlet pressure was controlled at 0.45 MPa, the outlet pressure at 0.25 MPa, and the system temperature at 32°C. Cross-flow filtration was performed to obtain a clear crude lysine solution. The bacterial cells retained by membrane separation were washed, activated, and concentrated before being used for inoculation of the next batch of fermentation.

[0035] (3) Post-processing Ion exchange: The crude lysine was adjusted to pH 4.0 with 12% hydrochloric acid solution, and then adsorbed and eluted using a cation exchange resin. The resin-to-fermentation broth volume ratio was 1:3.0. Crystallization and drying: The eluent obtained from ion exchange was concentrated to 600 g / L under vacuum at 65°C. The concentrate was then cooled to 12°C at a programmed rate of 1.2°C / h to crystallize. The crystals were then centrifuged to obtain wet crystals. After drying, high-purity lysine was obtained.

[0036] The lysine product prepared in this example has a purity of 98.8%.

[0037] Fermentation performance: Using glucose in the fermentation medium as the substrate, the sugar-acid conversion rate was calculated by detecting the lysine concentration in the fermentation broth at the fermentation endpoint. In this example, the sugar-acid conversion rate of lysine fermentation was 66.0%; the viscosity of the fermentation broth at the fermentation endpoint was 5.0 mPa·s. Membrane separation performance: Under the operating conditions of the membrane separation step in this embodiment, the flux decline rate of the surface-modified membrane was 13.9% after 4 hours of continuous operation; After 4 hours of continuous operation, the surface-modified membrane was rinsed for 30 minutes each with 1.0 wt% NaOH solution and 1.0 wt% HCl solution, and then rinsed with water until the pH of the rinsing solution was 7.0. The pure water flux recovery rate of the membrane was 96.9%.

[0038] Example 4 A fermentation-membrane separation coupled production method for high-purity lysine includes the following steps: (1) Fermentation Activation culture: Commercially available Corynebacterium glutamicum was inoculated into activation medium and cultured at 32°C for 23 hours. The activation medium consisted of: glucose 11 g / L, peptone 11 g / L, ammonium sulfate 10 g / L, KH₂PO₄ 2.4 g / L, sodium chloride 2.5 g / L, pH 6.8.

[0039] Seed culture: The activated bacterial solution was inoculated into the seed culture medium at an inoculation rate of 5.5% (v / v) and cultured at 30℃ for 19 h. The seed culture medium consisted of: glucose 85 g / L, ammonium sulfate 10 g / L, corn steep liquor powder 20 g / L, KH2PO4 2.5 g / L, MgSO4·7H2O 1.1 g / L, pH 6.8.

[0040] Fermentation culture: The seed culture was inoculated into the fermentation medium at an inoculation rate of 6.5% (v / v) and fermented at 34°C for 28 h. At 17 h of fermentation, N-(4-aminobutyl)acetamide was added at a concentration of 0.3 g / L. The fermentation medium consisted of: glucose 65 g / L, ammonium sulfate 15 g / L, corn steep liquor powder 36 g / L, KH₂PO₄ 3.2 g / L, MgSO₄·7H₂O 1.6 g / L, and pH 6.8.

[0041] (2) Membrane separation After fermentation, the fermentation broth was directly introduced into a membrane separation system composed of the surface-modified membrane prepared in Example 1. The inlet pressure was controlled at 0.5 MPa, the outlet pressure at 0.3 MPa, and the system temperature at 35°C for cross-flow filtration to obtain a clear crude lysine solution. The bacterial cells retained by membrane separation were washed, activated, and concentrated before being used for inoculation of the next batch of fermentation.

[0042] (3) Post-processing Ion exchange: The crude lysine was adjusted to pH 3.5 with 15% hydrochloric acid solution, and then adsorbed and eluted through a cation exchange resin with a resin-to-fermentation broth volume ratio of 1:2.8.

[0043] Crystallization and drying: The eluent obtained from ion exchange was concentrated to 650 g / L under vacuum at 70°C. The concentrate was then cooled to 10°C at a programmed rate of 1.5°C / h to crystallize. The crystals were then centrifuged to obtain wet crystals. After drying, high-purity lysine was obtained.

[0044] The lysine product prepared in this example has a purity of 98.6%.

[0045] Fermentation performance: Using glucose in the fermentation medium as the substrate, the sugar-acid conversion rate was calculated by detecting the lysine concentration in the fermentation broth at the fermentation endpoint. In this example, the sugar-acid conversion rate of lysine fermentation was 65.5%; the viscosity of the fermentation broth at the fermentation endpoint was 5.3 mPa·s. Membrane separation performance: Under the operating conditions of the membrane separation step in this embodiment, the flux decline rate of the surface-modified membrane was 14.1% after 4 hours of continuous operation. After 4 hours of continuous operation, the surface-modified membrane was rinsed for 30 minutes each with 1.0 wt% NaOH solution and 1.0 wt% HCl solution, and then rinsed with water until the pH of the rinsing solution was 7.0. The pure water flux recovery rate of the membrane was 96.3%.

[0046] Comparative Example 2 Comparative Example 2 uses the fermentation-membrane separation coupled production method of high-purity lysine described in Example 3. The difference is that in the membrane separation step, the surface-modified membrane prepared in Comparative Example 1 is used, while the other steps are the same.

[0047] The purity of the lysine product prepared in this comparative example is 97.9%.

[0048] Membrane separation performance: Under the operating conditions of the membrane separation step in the comparative example, the flux decline rate of the surface-modified membrane was 21.6% after 4 hours of continuous operation; After 4 hours of continuous operation, the surface-modified membrane was rinsed for 30 minutes each with 1.0 wt% NaOH solution and 1.0 wt% HCl solution, and then rinsed with water until the pH of the rinsing solution was 7.0. The pure water flux recovery rate of the membrane was measured to be 84.7%.

[0049] Comparative Example 3 Comparative Example 3 uses the fermentation-membrane separation coupled production method of high-purity lysine described in Example 3. The difference is that in the membrane separation step, an unmodified ceramic membrane is used to replace the surface-modified membrane. The ceramic membrane has ZrO2 as its composition and a pore size of 50 nm. All other steps are the same.

[0050] The purity of the lysine product prepared in this comparative example was 97.2%.

[0051] Membrane separation performance: Under the operating conditions of the membrane separation step in this embodiment, the flux decline rate of the surface-modified membrane was 38.5% after 4 hours of continuous operation; After 4 hours of continuous operation, the surface-modified membrane was rinsed for 30 minutes each with 1.0 wt% NaOH solution and 1.0 wt% HCl solution, and then rinsed with water until the pH of the rinsing solution was 7.0. The pure water flux recovery rate of the membrane was 62.1%.

[0052] Comparative Example 4 Comparative Example 4 uses the fermentation-membrane separation coupled production method of high-purity lysine described in Example 3. The difference is that the addition of N-(4-aminobutyl)acetamide is omitted in the fermentation culture step, while the other steps are the same.

[0053] The purity of the lysine product prepared in this comparative example is 97.8%.

[0054] Fermentation performance: Using glucose in the fermentation medium as the substrate, the sugar-acid conversion rate was calculated by detecting the lysine concentration in the fermentation broth at the fermentation endpoint. In this example, the sugar-acid conversion rate of lysine fermentation was 63.8%; the viscosity of the fermentation broth at the fermentation endpoint was 6.2 mPa·s. Membrane separation performance: Under the operating conditions of the membrane separation step in this embodiment, the flux decline rate of the surface-modified membrane was 17.8% after 4 hours of continuous operation; After 4 hours of continuous operation, the surface-modified membrane was rinsed for 30 minutes each with 1.0 wt% NaOH solution and 1.0 wt% HCl solution, and then rinsed with water until the pH of the rinsing solution was 7.0. The pure water flux recovery rate of the membrane was 94.5%.

[0055] Comparing Comparative Example 2 and Example 3, the analysis shows that, under the condition that the fermentation process is exactly the same, the membrane performance of Comparative Example 2, which uses a single molecular weight grafted membrane, is significantly worse than that of Example 3, which uses a double molecular weight grafted membrane. The 4-hour flux decay rate of Example 3 was only 13.9%, far lower than the 21.6% of Comparative Example 2. This demonstrates that the dual molecular weight synergistic graft layer has a stronger ability to maintain high flux during long-term operation and superior antifouling kinetics. The pure water flux recovery rate of Example 3 was as high as 96.9%, while that of Comparative Example 2 was only 84.7%. This indicates that the contaminants attached to the dual molecular weight graft layer have weaker binding force and are easier to clean, reducing the amount of chemical cleaning agents used and the damage to the membrane.

[0056] Comparing Comparative Example 4 and Example 3, the analysis shows that, with the membrane system being exactly the same, Example 3, with the addition of N-(4-aminobutyl)acetamide, is superior to Comparative Example 4, which did not add N-(4-aminobutyl)acetamide, in both fermentation yield and membrane fouling control. The lysine yield in Example 3 reached 66.0 g / L, significantly higher than the 63.8 g / L in Comparative Example 4, indicating that the additive effectively promoted the flow of carbon to lysine synthesis. Regarding membrane separation performance, the flux decline rate of Comparative Example 4 was 17.8%, and the pure water flux recovery rate was 94.5%, both lower than that of Example 3. Combined with the increased viscosity of the fermentation broth in Comparative Example 4, this may be because the addition of N-(4-aminobutyl)acetamide stabilized the cell membrane structure, reducing the release of sticky intracellular substances such as nucleic acids and proteins due to cell breakage, thus reducing the membrane fouling load at the source. Due to the reduced fouling source, the membrane flux recovery rate of Comparative Example 4 was still better than that of Comparative Example 2, but not as good as that of Example 3 with the complete scheme, indicating that the intervention of adding N-(4-aminobutyl)acetamide during the fermentation stage can improve the downstream separation effect.

[0057] Unless otherwise specified, all percentages mentioned in this application are percentages by mass.

[0058] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fermentation-membrane separation coupled production method for high-purity lysine, characterized in that, The method includes the following steps: Fermentation: In lysine fermentation medium, inoculate with Corynebacterium glutamicum seed culture and ferment at 32-34℃ for 28-30h. Add N-(4-aminobutyl)acetamide after 17-19h of fermentation. Membrane separation: The fermentation broth is cross-flow filtered using a surface-modified membrane with an inlet pressure of 0.4-0.5 MPa, an outlet pressure of 0.2-0.3 MPa, and a temperature controlled at 30-35℃. Crude lysine is obtained after membrane separation. Post-processing: The crude lysine product is subjected to ion exchange and crystallization to obtain a high-purity lysine product; The method for preparing the surface-modified film includes the following steps: film surface activation, silanization, and polymer grafting; Membrane surface activation: The ceramic membrane was treated with H2O2 solution, and after cleaning and drying, an activated membrane was obtained. Silanization: The activated membrane is immersed in a solution of γ-glycidyl oxypropyltrimethoxysilane and reacted at 58-62℃ for 4.8-5.2 hours. Then it is ultrasonically cleaned with ethanol 2-3 times and dried to obtain the silanized membrane. Polymer grafting: The silanized film is immersed in a poly(2-hydroxypropyl)methacrylamide solution, boron trifluoride diethyl ether is added, and the reaction is carried out at 68-72℃ for 3.8-4.2 hours; it is then ultrasonically cleaned in water at 40-45℃ 3-4 times, and dried at 45-50℃ for 11-13 hours to obtain a surface-modified film. The preparation of the poly(2-hydroxypropyl)methacrylamide solution involves dissolving low molecular weight poly(2-hydroxypropyl)methacrylamide and high molecular weight poly(2-hydroxypropyl)methacrylamide in water, wherein the molar ratio of low molecular weight poly(2-hydroxypropyl)methacrylamide to high molecular weight poly(2-hydroxypropyl)methacrylamide is 87-89:11-13, and the total concentration is 0.8-1 mol / L.

2. The fermentation-membrane separation coupled production method for high-purity lysine according to claim 1, characterized in that, The preparation of the Corynebacterium glutamicum seed culture medium includes activation culture and seed culture; The activation culture method is as follows: Corynebacterium glutamicum is inoculated into activation culture medium and cultured at 30-32℃ for 23-25 ​​hours. The Corynebacterium glutamicum was purchased from the market. The activated culture medium consists of 9-11 g / L glucose, 9-11 g / L peptone, 8-10 g / L ammonium sulfate, 2.0-2.4 g / L KH2PO4, 2-2.5 g / L sodium chloride, and has a pH of 6.8-7.

2. The seed culture method is as follows: the activated bacterial solution is inoculated into the seed culture medium at an inoculation amount of 4.5-5.5% by volume for seed culture, the culture temperature is 28-30℃, the culture time is 19-21h, and the seed culture solution is obtained after the culture is completed. The seed culture medium consists of 75-85 g / L glucose, 8-10 g / L ammonium sulfate, 15-20 g / L corn steep liquor powder, 1.5-2.5 g / L KH2PO4, 0.9-1.1 g / L MgSO4·7H2O, and a pH of 6.8-7.

2.

3. The fermentation-membrane separation coupled production method for high-purity lysine according to claim 1, characterized in that, The amount of N-(4-aminobutyl)acetamide added is 0.2-0.3 g / L.

4. The fermentation-membrane separation coupled production method for high-purity lysine according to claim 1, characterized in that, The fermentation medium consists of 55-65 g / L glucose, 10-15 g / L ammonium sulfate, 34-36 g / L corn steep liquor powder, 2.8-3.2 g / L KH2PO4, 1.4-1.6 g / L MgSO4·7H2O, and a pH of 6.8-7.

2. In the fermentation step, the amount of seed culture medium added is 5.5-6.5% of the volume of the fermentation medium.

5. The fermentation-membrane separation coupled production method for high-purity lysine according to claim 1, characterized in that, In the ion exchange step, the resin type is cation exchange resin, and the pH of crude lysine is adjusted to 3.5-4.5 with 10-15% hydrochloric acid solution. The volume percentage of resin to fermentation broth is 1:2.8-3.

2. The crystallization method is as follows: the eluent obtained by ion exchange is concentrated to 550-650 g / L at 60-70℃, and then the concentrated solution is cooled to 10-15℃ at 1-1.5℃ / h to obtain wet crystals. The wet crystals are dried to obtain the lysine product.

6. The fermentation-membrane separation coupled production method for high-purity lysine according to claim 1, characterized in that, In the preparation steps of the surface-modified film, The ceramic membrane is composed of ZrO2 and has a pore size of 40-60 nm.

7. The fermentation-membrane separation coupled production method for high-purity lysine according to claim 1, characterized in that, In the preparation steps of the surface-modified film, The concentration of the H2O2 solution is 3-4 wt%; The temperature for treating the ceramic membrane with the H2O2 solution is 38-42℃, and the treatment time is 30-35 minutes.

8. The fermentation-membrane separation coupled production method for high-purity lysine according to claim 1, characterized in that, In the preparation steps of the surface-modified film, The solvent for the γ-glycidyl etheroxypropyltrimethoxysilane solution is an ethanol-water mixture with a concentration of 1.5-2 wt%. The mass ratio of ethanol to water in the ethanol-water mixture is 93-96:4-7.

9. The fermentation-membrane separation coupled production method for high-purity lysine according to claim 1, characterized in that, In the preparation steps of the surface-modified film, The low molecular weight poly(N-(2-hydroxypropyl)methacrylamide) has a molecular weight of 800-1200; The high molecular weight poly(N-(2-hydroxypropyl)methacrylamide) has a molecular weight of 25,000-35,000; The mass ratio of boron trifluoride ether to poly(N-(2-hydroxypropyl)methacrylamide) solution is 0.4-0.6:100.