Process for optimizing nitrogen source in threonine fermentation

Abstract

The invention belongs to the technical field of biology, and discloses a process for optimizing a nitrogen source in threonine fermentation, a nitrogen source composition is used, the nitrogen source composition comprises corn steep liquor and ammonium sulfate, the corn steep liquor accounts for 65-75% of the total mass of the nitrogen source, and the ammonium sulfate accounts for 25-35% of the total mass of the nitrogen source. According to the invention, a composite nitrogen source system based on corn steep liquor-ammonium sulfate is established, the optimal ratio range is determined through a large number of experiments, the fermentation cost is reduced, and the yield and conversion rate of threonine are improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a process for optimizing nitrogen sources in threonine fermentation. Background Technology

[0002] Currently, soybean meal and yeast extract are the main organic nitrogen sources used in threonine fermentation production. This traditional method has two significant drawbacks: first, the raw material prices are high and fluctuate greatly, making it difficult to control production costs; second, the quality of the raw materials is unstable, directly affecting the stability of the fermentation process and the consistency of the product. These problems severely restrict the large-scale development and economic benefits of threonine production.

[0003] Nitrogen source optimization is particularly important in actual production during threonine fermentation, as it not only determines the fermentation cost but also affects the yield and conversion rate of threonine. Currently, the main technological innovations in nitrogen source optimization focus on three aspects: compound nitrogen source formulation design, nitrogen source feed control strategy, and development of novel nitrogen sources.

[0004] Regarding the formulation of compound nitrogen sources, patent document CN110923273A proposes an innovative process for preparing corn husk hydrolysate. Through ultrasonic-assisted hydrochloric acid hydrolysis and synergistic effects of microorganisms, the complete degradation of proteins and cellulose in corn husks is achieved. The resulting nitrogen source is rich in peptides and amino acids, increasing threonine yield by approximately 20%. This patent details the preparation steps of the hydrolysate, including key process parameters such as pretreatment, ultrasonic-assisted hydrolysis, microbial hydrolysis, cell hydrolysis, and concentrated steam treatment. Regarding the nitrogen source feed control strategy, patent document CN102304553A employs a two-stage feeding method: the first stage involves exponential feeding with the target of optimal specific production rate, and the second stage involves constant-rate feeding with the target of maximum production rate. L-Isoleucine plays a key role as the nitrogen source component, with its feeding rate precisely controlled at 1.5 mg / L. - ¹·h - ¹ This precise control significantly improves nitrogen source utilization efficiency. Regarding the development of novel nitrogen sources, patent document CN118853438A innovatively combines corn steep liquor and soybean meal hydrolysate in a specific ratio, along with a carbon source combination of fructose syrup and glucose, achieving the dual effect of reduced byproduct accumulation and increased conversion rate. This nitrogen source combination is suitable for the fermentation characteristics of Escherichia coli strain MHZ-0215-2. Summary of the Invention

[0005] This invention provides an optimized nitrogen source scheme for threonine fermentation, with the aim of reducing fermentation costs and increasing threonine yield and conversion rate.

[0006] The present invention is achieved through the following technical solution.

[0007] A nitrogen source composition for threonine fermentation, the nitrogen source composition comprising corn steep liquor and ammonium sulfate, wherein the corn steep liquor accounts for 65-75% of the total nitrogen source mass and the ammonium sulfate accounts for 25-35%.

[0008] Preferably, the corn steep liquor accounts for 70% of the total nitrogen source mass, and ammonium sulfate accounts for 30%.

[0009] More preferably, the amount of corn steep liquor added to the fermentation medium is 35 g / L, and the amount of ammonium sulfate is 15 g / L.

[0010] Preferably, the corn syrup has a Baume degree of 18-22°Bé.

[0011] As another technical solution of the present invention The invention relates to a threonine fermentation process using the aforementioned nitrogen source composition, characterized in that the temperature in the fermentation process is controlled and adjusted in stages between 33-36°C.

[0012] Preferably, the pH is controlled within the range of 7.0 ± 0.2 during the fermentation process.

[0013] Preferably, the fermentation medium used in the fermentation process is: corn steep liquor (70°Bé) 35 g / L, ammonium sulfate 15 g / L, glucose 100 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.015 g / L, pH natural.

[0014] Preferably, the fermentation process is divided into three stages of temperature control: initial stage (0-12h) 34±0.5℃, middle stage (12-36h) 36±0.5℃, and later stage (36h to 48h) 33±0.5℃. Furthermore, the fermentation process also includes a seed culture step.

[0015] Furthermore, the seed culture includes the following steps: The engineered *E. coli* strain was streaked onto an agar slant and cultured for 12 hours. Afterward, it was transferred to a secondary agar slant and cultured for another 12 hours. The activated strain was then transferred to a seed culture medium for further cultivation. The pH was controlled at 7.0, the temperature at 37℃, and the dissolved oxygen at 30%. The culture was continued until the OD600 reached 3-3.5. The seed culture medium consisted of: glucose 25 g / L, yeast extract 5 g / L, peptone 4 g / L, citric acid 2 g / L, KH₂PO₄ 0.5 g / L, MgSO₄·7H₂O 0.1 g / L, FeSO₄·7H₂O 20 mg / L, MnSO₄ 10 mg / L, and VB1, VB3, VB5, and VB12 each 2 mg / L.

[0016] The present invention is achieved through the following technical solution.

[0017] Through extensive experimental research, this invention has found that corn steep liquor, as an alternative organic nitrogen source, has the following advantages: it is rich in nutrients and has a balanced ratio, containing a variety of amino acids, vitamins and growth factors; its price is relatively stable and lower than that of traditional nitrogen sources, which can reduce raw material costs by about 15-20%; its industrial production has a high degree of standardization, with small quality differences between different batches; and it has good solubility and fermentation adaptability, which is conducive to the rapid absorption and utilization by microorganisms.

[0018] This study is the first to systematically verify the feasibility of using corn steep liquor to completely replace traditional soybean meal as a nitrogen source in threonine fermentation, solving the industry problem of high and unstable raw material prices. A fermentation control strategy tailored to the characteristics of corn steep liquor was developed, particularly segmented temperature control and metabolic state-based feeding technology. Without increasing equipment investment, simultaneous improvements in yield and conversion rate were achieved through nitrogen source optimization. This technical solution is applicable to fermentation production at different scales, demonstrating good stability and repeatability from pilot-scale to industrial-scale production. As a byproduct of corn starch processing, the utilization of corn steep liquor aligns with the concept of a circular economy, reducing the pressure on land resources from soybean meal production. Ammonium sulfate, being inexpensive, effectively meets the nitrogen source requirements of strains at different growth stages when used in combination with corn steep liquor.

[0019] The core innovation of this invention lies in establishing a composite nitrogen source system based on corn steep liquor and ammonium sulfate, and determining the optimal ratio range through numerous experiments: Organic nitrogen source components: The proportion of corn steep liquor in the total nitrogen source is 65-75% (w / w), preferably 70%. The Baume degree of corn steep liquor is controlled within the range of 18-22±0.5°Bé to ensure stable nutrient concentration.

[0020] Inorganic nitrogen source components: Ammonium sulfate accounts for 25-35% (w / w) of the total nitrogen source, preferably 30%. This is dynamically adjusted during the later stages of fermentation based on changes in residual sugar concentration and pH.

[0021] Carbon source component: glucose 80-100g / L.

[0022] Micronutrient supplementation: Add 0.05-0.1 g / L of MgSO4·7H2O, 0.01-0.03 g / L of MnSO4·H2O, and 0.005-0.015 g / L of FeSO4·7H2O to the basic formula to compensate for the lack of micronutrients in corn steep liquor. Detailed Implementation

[0023] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The products and methods of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the products and methods described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention. To further understand this invention, the following detailed description is provided in conjunction with embodiments. Example 1

[0024] The engineered Escherichia coli producing threonine was streaked onto PDA slant medium and cultured for 12 hours. The culture was then transferred to a PDA slant and continued for another 12 hours. The activated strain was then transferred to seed culture medium and cultured at a pH of 7.0, a constant temperature of 37°C, and dissolved oxygen of 30% until the OD600 reached 3.5. Seed culture medium composition: glucose 25g / L, yeast extract 5g / L, peptone 4g / L, citric acid 2g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.1g / L, FeSO4·7H2O 20mg / L, MnSO4 10mg / L, VB1, VB3, VB5, and VB12 2mg / L each.

[0025] A fermentation process verification experiment was conducted in a 5L fermenter, with the following specific parameters: Fermentation medium components: corn steep liquor (70°Bé) 35 g / L, ammonium sulfate 15 g / L, glucose 100 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.015 g / L, pH natural.

[0026] Temperature control: The fermentation time was 48 hours, the inoculum size was 8% (v / v), and the fermentation process was divided into three stages with temperature control: initial stage (0-12 hours) 34±0.5℃, middle stage (12-36 hours) 36±0.5℃, and late stage (36 hours to 48 hours) 33±0.5℃.

[0027] pH and residual sugar control: The pH is controlled within the range of 7.0±0.2 by automatic ammonia addition throughout the process. The residual sugar is maintained within the range of 5-10g / L by a feeding strategy based on the residual sugar concentration.

[0028] Dissolved oxygen control: Dissolved oxygen is maintained at 30% saturation by adjusting the stirring speed and aeration rate.

[0029] Under the above fermentation system, the effects of different nitrogen source ratios on fermentation acid production and conversion rate were verified. See Table 1 for details. Table 1

[0030] Example 2 The engineered Escherichia coli producing threonine was streaked onto PDA slant medium and cultured for 12 hours. The culture was then transferred to a PDA slant and continued for another 12 hours. The activated strain was then transferred to seed culture medium and cultured at a pH of 6.8, a constant temperature of 37°C, and dissolved oxygen at 35%, until the OD600 reached 3. Seed culture medium composition: glucose 25g / L, yeast extract 5g / L, peptone 4g / L, citric acid 2g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.1g / L, FeSO4·7H2O 20mg / L, MnSO4 10mg / L, VB1, VB3, VB5, and VB12 2mg / L each.

[0031] A fermentation process verification experiment was conducted in a 5L fermenter, with the following specific parameters: Fermentation medium components: corn steep liquor (70°Bé) 35 g / L, ammonium sulfate 15 g / L, glucose 100 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.015 g / L, pH natural.

[0032] Temperature control: The fermentation time was 48 hours, the inoculum size was 10% (v / v), and the fermentation process was divided into three stages of temperature control: initial stage (0-12 hours) 34±0.5℃, middle stage (12-36 hours) 36±0.5℃, and late stage (36 hours to 48 hours) 33±0.5℃.

[0033] pH control: The pH is controlled within the range of 7.0±0.2 by automatically adding ammonia water throughout the process.

[0034] Dissolved oxygen control: Dissolved oxygen is maintained at 25% saturation by adjusting the stirring speed and aeration rate.

[0035] Example 3 Pilot-scale production: Scale-up verification was carried out in a 500L fermenter using the same process parameters. The results are as follows: final threonine yield: 153 g / L, sugar-acid conversion rate: 62.5%, batch-to-batch variation coefficient <3%, which is significantly better than the traditional method.

[0036] Economic benefit analysis, calculated based on an annual production scale of 10,000 tons of threonine: Raw material cost savings: Corn steep liquor is about 2,000 yuan / ton cheaper than soybean meal, resulting in annual cost savings of about 7 million yuan; Increased conversion rate leads to improved profitability: The conversion rate increased from 55% to 62%, resulting in approximately a 10% increase in production with the same amount of raw material input. Reduced energy consumption: The fermentation cycle is shortened, and the energy consumption per unit product is reduced by 15%.

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the disclosed technical content without departing from the scope of the technical solution of the present invention, resulting in equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of the technical solution of the present invention.

Claims

1. A nitrogen source composition for threonine fermentation, characterized in that, The nitrogen source composition comprises corn steep liquor and ammonium sulfate, wherein the corn steep liquor accounts for 65-75% of the total nitrogen source mass and the ammonium sulfate accounts for 25-35%.

2. The nitrogen source composition according to claim 1, characterized in that, The corn steep liquor accounts for 70% of the total nitrogen source, and ammonium sulfate accounts for 30%.

3. The nitrogen source composition according to claim 1 or 2, characterized in that, The amount of corn steep liquor added to the fermentation medium is 35 g / L, and the amount of ammonium sulfate is 15 g / L.

4. The nitrogen source composition according to claim 1 or 2, characterized in that, The corn steep liquor has a Baume degree of 18-22°Bé.

5. A threonine fermentation process, employing the nitrogen source composition according to any one of claims 1-4, characterized in that, The fermentation process involves controlling the temperature in stages between 33-36℃.

6. The process according to claim 5, characterized in that, The pH is controlled within the range of 7.0 ± 0.2 during the fermentation process.

7. The process according to claim 5, characterized in that, The fermentation medium used in the fermentation process is: corn steep liquor (70°Bé) 35 g / L, ammonium sulfate 15 g / L, glucose 100 g / L, MgSO4·7H2O 0.1 g / L, MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.015 g / L, pH natural.

8. The process according to claim 5, characterized in that, The fermentation process is divided into three stages of temperature control: initial stage (0-12h) 34±0.5℃, middle stage (12-36h) 36±0.5℃, and late stage (36h to 48h) 33±0.5℃.

9. The process according to claim 5, characterized in that, The fermentation process also includes a seed culture step.

10. The process according to claim 9, characterized in that, The seed culture includes the following steps: Escherichia coli engineered strains are streaked onto an agar slant, cultured for 12 hours, then transferred to a secondary agar slant for another 12 hours. The activated strain is then transferred to a seed culture medium for further culture, maintaining pH at 7.0, temperature at 37℃, and dissolved oxygen at 30%, until the OD600 reaches 3-3.

5. The seed culture medium consists of: glucose 25 g / L, yeast extract 5 g / L, peptone 4 g / L, citric acid 2 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 0.1 g / L, FeSO4·7H2O 20 mg / L, MnSO4 10 mg / L, and VB1, VB3, VB5, and VB12 each 2 mg / L.

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