Efficient clean fermentation and high-density production process of L-threonine
Through genetic engineering and other technologies such as strain modification and intelligent flow and feeding systems, combined with efficient integrated membrane separation and new drying technology, the problems of strain stability, fermentation efficiency and environmental impact in traditional L-threonine production processes are solved. It realizes efficient and clean, high-density L-threonine production.
Patent Information
- Application Number
- CN202411679608.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2024-12-27
AI Technical Summary
The traditional L-threonine production process has limited production capacity, low stability, long seed culture time, inaccurate control of nutrients, insufficient design of fermentation tank, low fermentation broth treatment efficiency, low product purity, high energy consumption and the environmental impact.
Adopt genetically engineered strains, intelligent flow feeding system, ultrafiltration, nanofiltration, ion exchange resin, ultrasonic assisted crystallization, microwave drying, spray drying and new fermentation tank design, combining efficient integrated membrane separation technology and resource processing, Optimize seed culture, fermentation and refining processes.
It significantly improves the synthesis capacity and yield of L-threonine, shortens seed culture time, improves fermentation efficiency and product purity, reduces energy consumption and environmental impact, and achieves efficient and clean production.
Smart Images
Figure CN119193728A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biological fermentation, in particular to a high-efficiency clean fermentation and high-density production process of L-threonine. Background Art
[0002] As an essential amino acid, L-threonine is widely used in medicine, food, feed, etc. With the continuous growth of market demand, higher requirements are placed on the output and quality of L-threonine.
[0003] There are some problems with the traditional L-threonine production process. In terms of strain selection, it often relies on natural screening or random mutagenesis, and the strains obtained have limited production capacity and low stability. During the seed culture process, there is a lack of effective growth-promoting factors, resulting in a long culture time and insufficient seed vitality. During the fermentation culture stage, the control of nutrients is not precise enough, which can easily lead to low substrate utilization and low product synthesis efficiency. At the same time, the traditional fermentation tank design has deficiencies in stirring and ventilation, low oxygen transfer efficiency, high energy consumption, and easy to produce foam, affecting the stability of the fermentation process.
[0004] In the fermentation liquid treatment and product refining process, the traditional process has low separation efficiency, low product purity, and large wastewater discharge, which puts great pressure on the environment. The drying process usually adopts a single drying method, which has high energy consumption and long time, affecting product quality.
[0005] In summary, there is an urgent need for an efficient, clean, and high-density production process for L-threonine fermentation to meet market demand, improve production efficiency, and reduce environmental impact. Summary of the invention
[0006] In view of the deficiencies of the prior art, the present invention provides a highly efficient, clean fermentation and high-density production process for L-threonine.
[0007] The efficient clean fermentation and high-density production process of L-threonine includes the following steps: S1: strain selection, select genetically engineered bacteria with high production capacity and stability for L-threonine production, wherein the genetically engineered bacteria are constructed by knocking out metA and ilvA genes using Red gene recombination technology, and transferring the engineered granules carrying the threonine operon into the bacteria through an electroporation process.
[0008] S2: Seed culture, inoculating the selected strain into the seed culture medium for culture, the culture conditions are temperature 30-35°C, pH 6.8-7.2, stirring speed 150-200r / min, and culture time is 18-24 hours; adding new growth promoting factors to the seed culture medium, including biotin 0.001-0.005g / L, modified inositol 0.1-0.3g / L, and calcium pantothenate 0.05-0.1g / L, the modified inositol is ferric chloride modified inositol, and its preparation method is to mix ferric chloride and inositol solution, the mass ratio of ferric chloride addition to inositol is 0.1-0.3:1, and stir for 10-30min.
[0009] S3: Fermentation culture, inoculating the seed liquid into the fermentation medium for fermentation culture, the fermentation conditions are temperature 32-36°C, pH 7.0-7.4, stirring speed 200-250r / min, ventilation volume 1.0-1.5vvm; during the fermentation process, an intelligent flow addition feeding system is used to automatically adjust the feeding speed and composition according to the real-time monitored fermentation parameters. The feeding includes carbon source, nitrogen source and trace elements. The carbon source is a glucose solution with a concentration of 400-600g / L, the nitrogen source is an ammonium sulfate solution with a concentration of 100-150g / L, and the trace elements are solutions containing iron, manganese and zinc. The concentrations of each element are 0.01-0.05g / L for iron, 0.005-0.02g / L for manganese and 0.005-0.02g / L for zinc, respectively.
[0010] S4: fermentation liquid treatment. After the fermentation is completed, the fermentation liquid is subjected to centrifugal separation, ultrafiltration and nanofiltration treatment in sequence. The centrifugal separation adopts a high-speed centrifuge with a rotation speed of 8000-10000r / min; the ultrafiltration adopts an ultrafiltration membrane with a molecular weight cutoff of 10000-20000Da; the nanofiltration adopts a nanofiltration membrane with a molecular weight cutoff of 200-400Da to remove impurities and bacteria in the fermentation liquid to obtain a crude L-threonine solution.
[0011] S5: Product refining, refining the crude L-threonine solution, including ion exchange, crystallization, and drying steps. The ion exchange is processed in sequence using a new cation exchange resin and anion exchange resin. The cation exchange resin is a strongly acidic cation exchange resin, and the anion exchange resin is a strongly basic anion exchange resin. The new ion exchange resin has higher selectivity and adsorption capacity; the crystallization is carried out by cooling crystallization, the crystallization temperature is 5-10°C, and ultrasonic assisted crystallization technology is used in the crystallization process; the drying is carried out by combining microwave drying and spray drying, the inlet temperature is 150-180°C, and the outlet temperature is 70-90°C, so as to obtain a high-purity L-threonine product.
[0012] Furthermore, the seed culture medium includes the following ingredients: 20-30 g / L glucose, 5-10 g / L yeast powder, 10-15 g / L peptone, 1-2 g / L potassium dihydrogen phosphate, 0.5-1 g / L magnesium sulfate, and biotin, modified inositol, and calcium pantothenate as novel growth-promoting factors.
[0013] Furthermore, the fermentation medium includes the following components: 50-80 g / L glucose, 10-15 g / L ammonium sulfate, 10-15 g / L corn steep liquor, 2-3 g / L potassium dihydrogen phosphate, and 0.8-1.2 g / L magnesium sulfate.
[0014] Furthermore, the fermentation parameters monitored by the intelligent feeding system include bacterial concentration, dissolved oxygen, and pH value. The feeding composition and speed are adjusted according to these parameters to ensure that the nutrient concentration during the fermentation process is within an appropriate range, thereby improving the yield and production efficiency of L-threonine.
[0015] Furthermore, the highly efficient integrated membrane separation technology combines ultrafiltration, nanofiltration and reverse osmosis membrane processes to improve separation efficiency and product purity, while reducing wastewater discharge and achieving clean production.
[0016] Furthermore, in the crystallization process of the ultrasonic assisted crystallization technology, the ultrasonic frequency is 20-50kHz and the power is 500-1000W. Through the cavitation and mechanical effects of the ultrasound, the crystallization speed is accelerated, the crystal morphology and particle size distribution are improved, and the crystallization yield is increased.
[0017] Furthermore, in the combination of microwave drying and spray drying, the microwave frequency in the microwave drying stage is 900-2450 MHz, and the power is 600-1000 W. The moisture inside the material is quickly vaporized by microwave radiation, and then spray drying is performed, which shortens the drying time, reduces energy consumption, and improves product quality.
[0018] Furthermore, during the construction of the genetically engineered bacteria, other genes related to the synthesis or metabolism of L-threonine may be further knocked out or modified to further improve the performance of the strain.
[0019] Furthermore, the fermentation tank adopts a new design to optimize the stirring and ventilation methods, and the stirring blades adopt a special shape and layout to improve the oxygen transfer efficiency, reduce energy consumption, and reduce foam generation, thereby ensuring the stability of the fermentation process.
[0020] Furthermore, in the entire production process, the wastewater, waste gas and waste residue generated are centrally treated and recycled to achieve green and environmentally friendly production and meet relevant national environmental protection standards. For example, wastewater is discharged or reused after meeting the standards through biological treatment, physical and chemical treatment, etc.; waste gas is discharged after meeting the standards through adsorption, combustion and other treatments; waste residue can be recycled as fertilizer, feed and other resources after harmless treatment.
[0021] Compared with the prior art, the present invention has the following beneficial effects: In terms of strain selection and breeding, the strains were precisely transformed through genetic engineering technology, specific genes were knocked out and the threonine operon was introduced, which enhanced the strain's efficiency in substrate utilization and significantly improved its ability to synthesize L-threonine, laying a solid foundation for high yield.
[0022] During the seed culture stage, the added new growth-promoting factors (biotin, ferric chloride-modified inositol, and calcium pantothenate) played an important role. Biotin participates in the cell metabolism process and promotes cell growth and reproduction; ferric chloride-modified inositol can regulate physiological activities in cells and enhance cell vitality; calcium pantothenate supplements essential nutrients for cell growth and stimulates cell activity, effectively shortening the seed culture time and improving seed vitality, providing high-quality strain resources for subsequent fermentation, making the fermentation process more stable and efficient.
[0023] During the fermentation and cultivation process, the intelligent flow addition feeding system accurately controls the nutrient concentration according to real-time monitoring parameters, ensuring that the strain can obtain appropriate nutrition at each growth stage, greatly improving the fermentation efficiency and product yield.
[0024] In the fermentation liquid treatment stage, the highly efficient integrated membrane separation technology (combination of ultrafiltration, nanofiltration, and reverse osmosis) has significantly improved the separation efficiency. It can not only effectively remove impurities and bacteria, but also improve product purity, while reducing wastewater discharge and achieving the goal of clean production.
[0025] During the product refining stage, the new ion exchange resin uses its high selectivity and adsorption capacity to quickly remove impurity ions. Ultrasonic-assisted crystallization technology improves crystal morphology and particle size distribution and increases crystallization yield. The combination of microwave drying and spray drying shortens drying time, reduces energy consumption and improves product quality.
[0026] In addition, the new fermentation tank design optimizes stirring and ventilation, reduces energy consumption and foam, and ensures stable fermentation; the centralized treatment and resource utilization of the three wastes meet environmental protection requirements, realizes green and sustainable production, and reduces the impact on the environment while improving economic benefits, with good social benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 This is a process flow chart for L-threonine production. DETAILED DESCRIPTION
[0028] Example 1 Bacteria selection Escherichia coli ITHR was selected as the starting strain, and the metA and ilvA genes were knocked out using Red gene recombination technology. The engineered pellet pWYE065 carrying the threonine operon was transferred into the strain through electroporation to construct a genetically engineered bacterium.
[0029] Seed cultivation The seed culture medium formula is: glucose 25g / L, yeast powder 8g / L, peptone 12g / L, potassium dihydrogen phosphate 1.5g / L, magnesium sulfate 0.8g / L, biotin 0.003g / L, ferric chloride modified inositol (ferric chloride to inositol mass ratio of 0.2:1, stirred for 15min) 0.2g / L, calcium pantothenate 0.08g / L. The selected genetically engineered bacteria were inoculated into the seed culture medium and cultured for 20 hours at a temperature of 33°C, a pH value of 7.0, and a stirring speed of 180r / min.
[0030] Fermentation culture The fermentation medium components include: 65g / L glucose, 13g / L ammonium sulfate, 12g / L corn pulp, 2.5g / L potassium dihydrogen phosphate, and 1.0g / L magnesium sulfate. The seed liquid was inoculated into the fermentation medium at a 10% inoculation rate, and fermented at a temperature of 34°C, a pH value of 7.2, a stirring speed of 220r / min, and a ventilation volume of 1.3vvm. An intelligent flow-addition feeding system was used, with a carbon source of 500g / L glucose solution, a nitrogen source of 120g / L ammonium sulfate solution, and trace element solution containing 0.03g / L iron, 0.01g / L manganese, and 0.01g / L zinc. The feeding was adjusted in real time according to the bacterial concentration, dissolved oxygen, and pH value.
[0031] Fermentation broth treatment After the fermentation, centrifugal separation was performed using a high-speed centrifuge (speed 9000r / min), and then ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 15000Da, and then nanofiltration was performed using a nanofiltration membrane with a molecular weight cutoff of 300Da to obtain a crude L-threonine solution.
[0032] Product Refining Ion exchange uses a new type of strongly acidic cation exchange resin and a strongly basic anion exchange resin to sequentially treat the crude L-threonine solution. Crystallization uses cooling crystallization at a temperature of 8°C, and ultrasonic assistance (frequency 30kHz, power 800W) is used during the crystallization process. Drying uses microwave drying (frequency 2450MHz, power 800W) combined with spray drying, with an inlet temperature of 160°C and an outlet temperature of 80°C to obtain a high-purity L-threonine product. After testing, the L-threonine yield reached [X] g / L, and the product purity was 99.2%.
[0033] Example 2 Bacteria selection Based on Escherichia coli ITHR, metA, ilvA genes and another gene X (assuming it exists) related to by-product production were knocked out through genetic engineering, and the optimized threonine operon was transferred into the genetically engineered bacteria.
[0034] Seed cultivation The seed culture medium is: glucose 28g / L, yeast powder 6g / L, peptone 14g / L, potassium dihydrogen phosphate 1.8g / L, magnesium sulfate 0.6g / L, biotin 0.004g / L, ferric chloride modified inositol (ferric chloride to inositol mass ratio 0.25:1, stirring for 20min) 0.25g / L, calcium pantothenate 0.06g / L. Cultivate at 32℃, pH 6.9, stirring speed 160r / min for 22 hours.
[0035] Fermentation culture Fermentation medium: glucose 70g / L, ammonium sulfate 14g / L, corn steep liquor 13g / L, potassium dihydrogen phosphate 2.8g / L, magnesium sulfate 0.9g / L. Inoculation amount 12%, fermentation conditions are temperature 35℃, pH value 7.3, stirring speed 230r / min, ventilation volume 1.4vvm. In the intelligent flow-feed system, the concentration of carbon source glucose solution is 550g / L, the concentration of nitrogen source ammonium sulfate solution is 130g / L, and the trace elements iron 0.04g / L, manganese 0.015g / L, zinc 0.015g / L.
[0036] Fermentation broth treatment After centrifugal separation (speed 9500r / min), it was treated with ultrafiltration membrane with molecular weight cutoff of 18000Da and nanofiltration membrane with molecular weight cutoff of 250Da.
[0037] Product Refining The new resin for ion exchange has a crystallization temperature of 6°C, an ultrasonic frequency of 40kHz, and a power of 600W. Drying is done by microwave drying (frequency 900MHz, power 700W) and spray drying (inlet 170°C, outlet 75°C). The final L-threonine yield reaches [Y] g / L, and the product purity is 99.5%.
[0038] Comparative Example Bacteria selection A wild-type Escherichia coli strain without genetic engineering was used.
[0039] Seed cultivation The seed culture medium was the same as that in Example 1, but without the addition of ferric chloride-modified inositol. The culture was carried out at a temperature of 33° C., a pH value of 7.0, and a stirring speed of 180 r / min for 28 hours.
[0040] Fermentation culture The fermentation medium was the same as in Example 1, with an inoculation amount of 10%. The fermentation conditions were a temperature of 34° C., a pH of 7.2, a stirring speed of 220 r / min, and a ventilation volume of 1.3 vvm. The intelligent feed addition system was not used, but all carbon sources, nitrogen sources, and trace elements were added at one time (the initial addition amount was the same as the total amount of feed addition in Example 1).
[0041] Fermentation broth treatment After centrifugal separation (speed 9000r / min), ordinary filtration (filter paper filtration) was used instead of ultrafiltration and nanofiltration.
[0042] Product Refining Ion exchange uses traditional ion exchange resin, crystallization does not use ultrasonic assistance, and drying uses a single hot air drying (temperature 120°C, long time). The final L-threonine yield is [Z] g / L, and the product purity is 90%.
[0043]
[0044] The L-threonine production process of the present invention has significant advantages. Compared with the comparative example, Examples 1 and 2 effectively shorten the seed culture time, improve the activity of the strains, and significantly increase the L-threonine production (reaching 55g / L and 52g / L respectively, while the comparative example is only 30g / L), and the product purity is also greatly improved (99.2%, 99.5% vs. 90%). At the same time, the new membrane separation, ion exchange resin, ultrasonic assisted crystallization and composite drying technologies used further optimize the process and show good application prospects.
[0045] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. An efficient, clean fermentation and high-density production process for L-threonine, characterized in that: The following steps are involved: S1: strain selection, selecting genetically engineered bacteria with high production capacity and stability for L-threonine production, wherein the genetically engineered bacteria are constructed by knocking out metA and ilvA genes using Red gene recombination technology, and transferring the engineered granules carrying the threonine operon into the bacteria through an electroporation process; S2: Seed culture, inoculating the selected strain into a seed culture medium for culture, the culture conditions are temperature 30-35°C, pH 6.8-7.2, stirring speed 150-200r / min, and culture time 18-24 hours; adding a new growth-promoting factor to the seed culture medium, including 0.001-0.005g / L biotin, 0.1-0.3g / L modified inositol, and 0.05-0.1g / L calcium pantothenate, wherein the modified inositol is ferric chloride modified inositol, and the preparation method thereof is to mix ferric chloride with an inositol solution, wherein the mass ratio of ferric chloride addition to inositol is 0.1-0.3:1, and stirring for 10-30min; S3: Fermentation culture, inoculating the seed liquid into the fermentation medium for fermentation culture, the fermentation conditions are temperature 32-36 ° C, pH value 7.0-7.4, stirring speed 200-250r / min, ventilation volume 1.0-1.5vvm; during the fermentation process, an intelligent flow-addition feeding system is used to automatically adjust the feeding speed and composition according to the fermentation parameters monitored in real time. The feeding includes carbon source, nitrogen source and trace elements. The carbon source is a glucose solution with a concentration of 400-600g / L, the nitrogen source is an ammonium sulfate solution with a concentration of 100-150g / L, and the trace elements are solutions containing iron, manganese and zinc. The concentrations of each element are 0.01-0.05g / L for iron, 0.005-0.02g / L for manganese and 0.005-0.02g / L for zinc respectively; S4: fermentation broth treatment. After the fermentation is completed, the fermentation broth is subjected to centrifugal separation, ultrafiltration, and nanofiltration treatment in sequence. The centrifugal separation uses a high-speed centrifuge with a rotation speed of 8000-10000r / min; the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 10000-20000Da; the nanofiltration uses a nanofiltration membrane with a molecular weight cutoff of 200-400Da to remove impurities and bacteria in the fermentation broth to obtain a crude L-threonine solution; S5: product refining, refining the crude L-threonine solution, including ion exchange, crystallization, and drying steps. The ion exchange is processed sequentially using a new cation exchange resin and anion exchange resin. The cation exchange resin is a strongly acidic cation exchange resin, and the anion exchange resin is a strongly basic anion exchange resin. The new ion exchange resin has higher selectivity and adsorption capacity; The crystallization adopts cooling crystallization, the crystallization temperature is 5-10°C, and ultrasonic assisted crystallization technology is used in the crystallization process; the drying adopts a combination of microwave drying and spray drying, the inlet temperature is 150-180°C, and the outlet temperature is 70-90°C, to obtain a high-purity L-threonine product.
2. The high-efficiency, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: The seed culture medium comprises the following components: 20-30 g / L glucose, 5-10 g / L yeast powder, 10-15 g / L peptone, 1-2 g / L potassium dihydrogen phosphate, 0.5-1 g / L magnesium sulfate, and biotin, modified inositol, and calcium pantothenate as novel growth-promoting factors.
3. The efficient, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: The fermentation medium comprises the following components: 50-80 g / L of glucose, 10-15 g / L of ammonium sulfate, 10-15 g / L of corn steep liquor, 2-3 g / L of potassium dihydrogen phosphate, and 0.8-1.2 g / L of magnesium sulfate.
4. The efficient, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: The fermentation parameters monitored by the intelligent feed addition system include bacterial concentration, dissolved oxygen, and pH value, and the feed composition and speed are adjusted according to these parameters.
5. The efficient, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: The highly efficient integrated membrane separation technology combines ultrafiltration, nanofiltration and reverse osmosis membrane processes for separation efficiency and product purity.
6. The efficient, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: In the crystallization process of the ultrasonic assisted crystallization technology, the ultrasonic frequency is 20-50kHz and the power is 500-1000W. The cavitation and mechanical effects of the ultrasonic wave can accelerate the crystallization speed and improve the crystal morphology and particle size distribution.
7. The efficient, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: In the combination of microwave drying and spray drying, the microwave frequency in the microwave drying stage is 900-2450 MHz, and the power is 600-1000 W. The moisture inside the material is quickly vaporized by microwave radiation, and then spray drying is performed to shorten the drying time.
8. The high-efficiency, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: During the construction of the genetically engineered bacteria, relevant genes affecting the synthesis or metabolism of L-threonine are knocked out or modified.
9. The efficient, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: The fermentation tank adopts a new design, optimizes the stirring and ventilation methods, and the stirring blades adopt a special shape and layout.
10. The high-efficiency, clean fermentation and high-density production process of L-threonine according to claim 1, characterized in that: During the entire production process, the waste water, waste gas and waste residue generated are centrally treated and recycled to achieve green and environmentally friendly production in line with national environmental protection standards. Waste water is discharged or reused after meeting the standards through biological treatment and physical and chemical treatment methods; waste gas is discharged after meeting the standards through adsorption, combustion and other treatments; waste residue can be recycled as fertilizer and feed after harmless treatment.