Method for recovering cytosine from cytosine crude product mother liquor

Abstract

The invention relates to the technical field of biochemical engineering, and discloses a method for recovering cytosine from cytosine crude product mother liquor, which comprises the following four steps: resin adsorption and elution, concentration and crude product crystallization, dissolution decoloration and fine product crystallization, and reverse osmosis deep treatment. The combined crude mother liquor is subjected to specific resin adsorption elution, vacuum concentration crystallization, activated carbon decoloration refining and reverse osmosis treatment, so that efficient recovery of cytosine and resource utilization of waste liquor are realized. According to the method, ion exchange resin is selected and matched with ammonia water elution, key process parameters are controlled, the total recovery rate of cytosine reaches 76.48%-78.13%, and the product purity is larger than or equal to 99.0% (the impurity content is smaller than or equal to 1.0%); after reverse osmosis treatment, purified water can be recycled and fermented, concentrated solute can be circularly adsorbed, resource waste and waste liquid discharge are reduced, the process operation is mild, the flow is simple, and the method is suitable for industrial recovery of cytosine and pyrimidine and purine similar structure nucleotide products and conforms to the green sustainable production concept.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, in particular to a method for recovering cytosine from cytosine crude mother liquor, which is especially suitable for the industrialized large-scale recovery production of nucleotide products. BACKGROUND

[0002] As a key base for synthesizing DNA and RNA in vivo, cytosine plays an important role in life activity regulation and physiological and biochemical processes, and is widely used in fine chemical industry and biological medicine, and has a large market demand.

[0003] In the industrialized production of cytosine, the mainstream process is based on microbial fermentation, and the fermentation broth needs to go through a series of conventional separation and purification steps of biological drugs to obtain the target product. In this process, due to the difference in separation characteristics of cytosine and impurities and the selectivity of the crystallization process, multiple mother liquors containing residual cytosine and various impurities will inevitably be produced, including crude mother liquor, secondary mother liquor and fine mother liquor. The crude mother liquor refers to the filtration mother liquor produced when the cytosine fermentation broth is subjected to primary treatment (such as centrifugal removal of bacterial bodies, preliminary impurity removal), and then subjected to first purification operation such as concentration and cooling crystallization to separate the cytosine crude product. The residual amount of cytosine in the crude mother liquor is relatively high, and it also contains complex impurities such as fermentation by-products (such as amino acids, polypeptides), salts, colloids and macromolecular organic substances, which is the core target liquid for cytosine recovery.

[0004] At present, the existing technology has the following problems: first, the crude mother liquor is directly discharged or discharged after simple treatment, resulting in a large amount of recyclable resources being lost and increasing the production cost; second, the traditional recovery process is complex, inefficient, and the recovery purity is not high, which is difficult to meet the needs of industrial production; third, the existing method may use a large amount of chemical reagents, which is easy to introduce new impurities, and a large amount of wastewater and waste residue are produced, which does not meet the green and environmental protection production concept. Therefore, it is of great practical significance to develop a method for recovering cytosine from cytosine crude mother liquor which is efficient, environmentally friendly and low cost. SUMMARY

[0005] The present application aims to overcome the defects of low recovery efficiency, insufficient product purity, serious environmental pollution and high production cost of cytosine crude mother liquor in the prior art, and to provide a green and environmentally friendly, high recovery rate, high purity and low cost method for recovering cytosine crude mother liquor, and to realize the recycling of waste liquid resources.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: A method for recovering cytosine from cytosine crude mother liquor, comprising the following steps: S1, resin adsorption and elution The combined crude mother liquor is adsorbed by a pretreated resin chromatographic column at a flow rate of 1-5 BV / h, and after saturation, eluted with 0.2-0.6 mol / L eluent at a flow rate of 0.5-2.5 BV / h, and the eluate is collected.

[0007] In this step, the preliminary efficient separation of cytosine and impurities in the crude mother liquor is realized through the selective adsorption of ion exchange resin. Among them: The 001x7 type and SP117 type cation exchange resin are suitable for the crude mother liquor in which cytosine exists in the form of cation, and the D217 type and SP217 type anion exchange resin are suitable for the crude mother liquor in which cytosine exists in the form of anion. The resin can be flexibly adapted according to the existing form of cytosine and the type of impurity ions in the crude mother liquor, to ensure high selective adsorption of cytosine; The eluent is preferably 0.3-0.5 mol / L ammonia water. Ammonia water has the characteristics of low toxicity, easy volatilization and no residue. It can be completely removed through subsequent vacuum concentration process, avoiding the introduction of new organic impurities, and the source of ammonia water is widely available and low in cost; The adsorption flow rate is controlled at 1-3 BV / h, which can ensure sufficient time for cytosine to combine with the active sites of the resin to achieve full adsorption, and also take into account the processing efficiency. The elution flow rate is controlled at 0.5-1.5 BV / h, which can ensure that the cytosine adsorbed on the resin is completely eluted, avoiding incomplete elution due to too fast flow rate, or prolonged production cycle due to too slow flow rate.

[0008] Resin pretreatment method: the resin is eluted with 4-6 BV of 5% hydrochloric acid solution at a flow rate of 2-3 BV / h, and then washed with 4-6 BV of purified water until the pH of the effluent is 3-4; then eluted with 4-6 BV of 5% sodium hydroxide solution at a flow rate of 2-3 BV / h, and then washed with 4-6 BV of purified water until the pH of the effluent is 7-8, ready for use.

[0009] S2, concentration and crude product crystallization The eluate is vacuum concentrated at 70-80℃ and -0.075 to -0.09 MPa to obtain a concentrated solution with a concentration of 30-50 g / L, cooled to 0-4℃ for crystallization, and incubated for 4-12 h. The filter cake is washed with 20%-50% (preferably 20%-30%) of 0-4℃ purified water based on the volume of the crystallization solution, and the wet crude product and secondary mother liquor are collected.

[0010] In this step, the preliminary enrichment and purification of cytosine are realized under mild conditions: Vacuum concentration technology is used to reduce the boiling point of the solution, and efficient concentration of the eluate is realized at a mild temperature of 70-80℃, avoiding the destruction of cytosine molecular structure caused by long-term high temperature, and ensuring the activity of the product; The concentration of the concentrated solution is controlled in the range of 30-50 g / L, which can ensure the efficient progress of the subsequent crystallization process and avoid the synchronous precipitation of impurities due to too high concentration, thereby affecting the purity of the crude product. The low temperature environment of 0-4℃ can reduce the solubility of cytosine and promote the formation of stable crystals, and the crystal growth time of 4-12h can make the crystals grow fully and reduce the impurities wrapped in the crystals. The filter cake is washed with 20%-30% 0-4℃ purified water (corresponding to the preferred range defined in claim 5), which can effectively remove the soluble impurities attached to the surface of the crystals while minimizing the loss of cytosine due to dissolution, thereby balancing the purification effect and recovery rate; the basic range of 20%-50% provides flexible adjustment space for industrial production, which can adapt to different washing needs according to the impurity content of the crude product.

[0011] S3, dissolution and decolorization and fine crystallization The wet crude product is dissolved according to the cytosine concentration of 20-50 g / L (preferably 30-40 g / L), 5-15% of activated carbon is added for decolorization for 30-60 min, the temperature is maintained at 85-95℃ during the decolorization process to prevent premature crystallization of the solution, and the decolorization solution is cooled to 0-4℃ while stirring for 2-8h for crystal growth, then the filter cake is washed with 20%-50% 0-4℃ purified water based on the volume of the crystallization solution, and the wet fine product is sent to a dryer for drying, and the fine product mother liquor is collected.

[0012] This step realizes the purification of cytosine through dissolution and recrystallization and activated carbon decolorization, which greatly improves the product purity: The solution is prepared according to the cytosine concentration of 30-40 g / L, which can ensure the stability and fluidity of the solution, avoid low concentration leading to low crystallization efficiency, or high concentration leading to premature precipitation of impurities; 5-8% of activated carbon is added according to the mass of cytosine, which has a large specific surface area and strong adsorption, can efficiently adsorb pigments, trace organic impurities and part of inorganic impurities in the solution, and the decolorization time is controlled in the range of 30-60 min, which can ensure the decolorization effect and avoid excessive adsorption of cytosine by activated carbon leading to low recovery rate; The temperature is maintained at 85-95℃ during the decolorization process to prevent premature crystallization of the solution, ensuring uniform and thorough decolorization; the filter cake is washed with 20%-50% 0-4℃ purified water based on the volume of the crystallization solution, which can effectively remove the trace impurities remaining on the surface of the crystals while minimizing the loss of cytosine due to excessive washing, thereby ensuring the recovery rate and purity of the product; the subsequent low-temperature crystallization and washing steps further remove residual impurities, and finally the purity of cytosine product is ≥99.0% (impurity content ≤1.0%).

[0013] S4, reverse osmosis deep treatment The secondary mother liquor, the refined mother liquor, and the wastewater generated from resin regeneration are combined and sent to a reverse osmosis unit. The reverse osmosis unit uses a polyamide composite reverse osmosis membrane (with a molecular weight cutoff of ≤200 Da at 90% rejection rate) and processes the water under a pressure of 0.6 to 0.8 MPa to recover the purified water that can be reused for fermentation and collect the concentrated solute. The concentrated solute is returned to step S1 for re-adsorption by the resin.

[0014] This step achieves the resource recycling and harmless treatment of waste liquid: The combined wastewater passes through a reverse osmosis membrane at a pressure of 0.6–0.8 MPa. The membrane module can remove impurities such as ions, organic matter, and suspended particles from the water, forming purified water with a conductivity of ≤60 μS / cm and a COD of ≤45 mg / L. The quality of this purified water meets the water requirements of the fermentation process and can be directly reused in fermentation production, significantly reducing the consumption of fresh water resources. The concentrated solute obtained by interception contains cytosine and other valuable components that have not been fully recovered. It is returned to step S1 for resin adsorption again to further improve resource utilization and increase the total recovery rate of cytosine to more than 76%. This step significantly reduces the amount of industrial wastewater discharged, lowers environmental treatment costs, and achieves a closed-loop recycling of wastewater.

[0015] Compared with the prior art, the present invention has the following significant advantages: (1) High resource utilization rate: Through the integrated process of "resin adsorption, crystallization purification and reverse osmosis circulation", the maximum recovery of cytosine and other valuable components in the crude mother liquor is achieved. The total recovery rate of cytosine reaches 76.48% to 78.13%, which significantly improves resource utilization and reduces production costs. (2) Excellent product purity: After multiple purification processes such as resin adsorption to remove impurities, activated carbon decolorization, and multiple crystallization washing, the product purity is ≥99%, and can reach up to 99.8%, with impurity content ≤0.2%, meeting the high standard requirements of biomedicine, fine chemical industry and other fields; (3) Green and environmentally friendly: Ammonia water is used as the eluent, leaving no residue and causing no pollution; reverse osmosis treatment realizes the closed loop of wastewater resource utilization, reducing wastewater discharge by more than 85% (taking Example 1 as an example, the traditional process requires direct discharge of 80L of resin regeneration wastewater + 2.22L of secondary mother liquor per batch + 1.62L of refined mother liquor per batch, totaling 83.84L; this invention combines the 83.84L of wastewater and treats it through reverse osmosis, recovering 74L of purified water to be directly reused in the fermentation process, and collecting 6L of concentrated solute to be returned to step S1 for recycling and adsorption, so there is no actual external wastewater discharge, and the wastewater reduction rate relative to the traditional process is (83.84-0) / 83.84×100%≈99.9%; in multiple batches of industrial production, a small amount of auxiliary wastewater is generated due to equipment cleaning, etc., and the average reduction rate of each example is ≥85%), significantly reducing the consumption of fresh water; the entire process does not use organic solvents and does not generate harmful waste residue, which is in line with the concept of green and sustainable development; (4) Strong industrial applicability: The process steps are simple and coherent, the operating conditions are mild and easy to control, the range of key parameters is wide, and it can be seamlessly connected with the existing cytosine fermentation production line without large-scale equipment modification. It has high processing efficiency and is suitable for large-scale industrial production. (5) Good process stability: By optimizing key parameters such as resin type, flow rate, temperature and concentration, the entire recycling process is ensured to be stable and reliable, with small fluctuations in product quality and a recovery rate of over 76%. Detailed Implementation

[0016] To make the technical solution of the present invention clearer and more explicit, the embodiments of the present invention will be further described in detail below through specific examples. Example

[0017] S1. Resin adsorption and elution: Take 7.5 L of the combined crude mother liquor (cytosine concentration in the crude mother liquor is 7.7 g / L) and pass it through a pretreated 001×7 type resin chromatography column at a flow rate of 3 BV / h for adsorption. After adsorption saturation, elute with 0.5 mol / L ammonia water at a flow rate of 1.0 BV / h and collect 11.34 L of eluent (cytosine concentration in the eluent is 4.88 g / L). S2. Concentration and Crude Product Crystallization: The eluent was concentrated under vacuum at 75℃ and -0.075MPa (during the concentration process, there was a slight mass loss due to ammonia evaporation and a small amount of cytosine adsorption on the inner wall of the equipment, with a loss rate ≤1.5%), yielding 1.82L of concentrate (cytosine concentration in the concentrate was 30.4g / L). The concentrate was cooled to 4℃ for crystallization and cultured for 12h. The crystals were then filtered using a centrifuge. The filter cake was washed with purified water at 0-4℃, accounting for 20% of the volume of the crystallization liquid, yielding 63g of wet crude product (moisture content of the wet crude product was 23.3%, and the cytosine concentration after drying was 95.2%). 2.22L of secondary mother liquor was collected (cytosine concentration in the secondary mother liquor was 3.71g / L). S3. Dissolution, Decolorization, and Refined Crystallization: Using purified water as the solvent, a solution was prepared at a cytosine concentration of 35 g / L. Activated carbon was added at 5% of the cytosine mass. The solution was decolorized at 85°C for 30 min. After decarbonization using a precision filter, the decolorized solution was cooled to 4°C while stirring. Crystallization was carried out for 3–4 h. The solution was then filtered using a centrifuge. The filter cake was washed with purified water at 0–4°C, accounting for 20% of the volume of the crystallization solution. The wet refined product was sent to a desiccator and dried at 60–80°C and 0.1 MPa (standard atmospheric pressure) until the moisture content was ≤0.5%, yielding 44.8 g of the finished product (finished product purity 99.2%, impurity content ≤0.8%). 1.62 L of refined mother liquor was collected (cytosine concentration in the mother liquor was 1.35 g / L). S4. Deep Reverse Osmosis Treatment: Multiple batches of secondary mother liquor, refined mother liquor, and 80L of resin regeneration wastewater are combined (the combined wastewater has a conductivity of 1450μS / cm and a COD of 850mg / L). The combined wastewater is sent to a reverse osmosis unit using a polyamide composite reverse osmosis membrane (with a molecular weight cutoff of ≤200Da at 90% rejection rate) and treated at a pressure of 0.6MPa. 74L of purified water is recovered (purified water has a conductivity of 50μS / cm and a COD of 40mg / L, which can be reused for fermentation). 6L of concentrated solute is collected and returned to step S1 for re-adsorption by the resin.

[0018] In this example, the overall recovery rate of cytosine was 77.58%. Example

[0019] S1. Resin adsorption and elution: Take 6.8 L of the combined crude mother liquor (cytosine concentration in the crude mother liquor is 7.85 g / L) and pass it through a pretreated SP117 resin chromatography column at a flow rate of 2 BV / h for adsorption. After adsorption saturation, elute with 0.5 mol / L ammonia water at a flow rate of 1.0 BV / h and collect 10.79 L of eluent (cytosine concentration in the eluent is 4.75 g / L). S2. Concentration and Crude Product Crystallization: The eluent was concentrated under vacuum at 77℃ and -0.08MPa (a small amount of mass loss occurred during the concentration process due to ammonia evaporation and a small amount of cytosine adsorbed on the inner wall of the equipment, with a loss rate ≤1.5%), yielding 1.70L of concentrate (cytosine concentration in the concentrate was 30.1g / L). The concentrate was cooled to 4℃ for crystallization and cultured for 12h. The crystals were then filtered using a centrifuge. The filter cake was washed with purified water at 0-4℃, accounting for 25% of the volume of the crystallization liquid, yielding 60.02g of wet crude product (moisture content of the wet crude product was 23%, and the cytosine concentration after drying was 94.8%). 2.05L of secondary mother liquor was collected (cytosine concentration in the secondary mother liquor was 3.43g / L). S3. Dissolution, Decolorization, and Refined Crystallization: Using purified water as the solvent, a solution was prepared at a cytosine concentration of 35 g / L. Activated carbon was added at 6% of the cytosine mass. The solution was decolorized at 90℃ for 40 min. After decarbonization using a precision filter, the decolorized solution was cooled to 4℃ while stirring, and crystallized for 4 h. The solution was then filtered using a centrifuge. The filter cake was washed with purified water at 0-4℃, accounting for 25% of the crystallization liquid volume. The wet refined product was sent to a desiccator and dried at 60-80℃ and 0.1 MPa (standard atmospheric pressure) until the moisture content was ≤0.5%, yielding 41.03 g of the finished product (finished product purity 99.5%, impurity content ≤0.5%). 1.59 L of refined mother liquor was collected (cytosine concentration in the mother liquor was 1.49 g / L). S4. Reverse Osmosis Deep Treatment: Multiple batches of secondary mother liquor, refined mother liquor, and 80L of resin regeneration wastewater are combined (the combined wastewater has a conductivity of 1650μS / cm, COD of 950mg / L, and a total volume of approximately 83.64L). This wastewater is then fed into a reverse osmosis unit using a polyamide composite reverse osmosis membrane (with a molecular weight cutoff of ≤200Da at 90% rejection rate) and treated at a pressure of 0.6MPa (due to membrane residue and a small amount of evaporation during the reverse osmosis process, there is a reasonable volume loss of ≤6%). 72.7L of purified water is recovered (the purified water has a conductivity of 60μS / cm, COD of 45mg / L, and can be reused for fermentation). 5.8L of concentrated solute is collected and returned to step S1 for re-adsorption by the resin.

[0020] In this example, the overall recovery rate of cytosine was 76.48%. Example

[0021] S1. Resin Adsorption and Elution: Take 8.5 L of the combined crude mother liquor (cytosine concentration in the crude mother liquor is 7.62 g / L) and pass it through a pretreated D217 resin chromatography column at a flow rate of 1.5 BV / h for adsorption. After adsorption saturation, elute with 0.3 mol / L ammonia water at a flow rate of 0.5 BV / h and collect 12.48 L of eluent (cytosine concentration in the eluent is 5.05 g / L). S2. Concentration and Crude Product Crystallization: The eluent was concentrated under vacuum at 75℃ and -0.075MPa (during the concentration process, there was a slight mass loss due to ammonia evaporation and a small amount of cytosine adsorption on the inner wall of the equipment, with a loss rate ≤1.5%), yielding 1.97L of concentrate (cytosine concentration in the concentrate was 31.62g / L). The concentrate was cooled to 4℃ for crystallization and cultured for 12h. The crystals were then filtered using a centrifuge, and the filter cake was washed with purified water at 0-4℃, accounting for 20% of the volume of the crystallization liquid, yielding 72.96g of wet crude product (moisture content of the wet crude product was 24.3%, and the cytosine concentration after drying was 95.9%). 2.36L of secondary mother liquor was collected (cytosine concentration in the secondary mother liquor was 3.70g / L). S3. Dissolution, Decolorization, and Refined Crystallization: Using purified water as a solvent, a solution was prepared at a cytosine concentration of 35 g / L. Activated carbon was added at 5% of the cytosine mass. Decolorization was carried out at 95°C for 30 min (within the 30-60 min range specified in claim 1). After decarbonization using a precision filter, the decolorized solution was cooled to 4°C while stirring, and crystallized for 3-4 h. The solution was then filtered using a centrifuge. The filter cake was washed with purified water at 0-4°C, accounting for 20% of the volume of the crystallization solution. The wet refined product was sent to a desiccator and dried at 60-80°C and 0.1 MPa (standard atmospheric pressure) until the moisture content was ≤0.5%, yielding 50.97 g of the finished product (finished product purity 99.8%, impurity content ≤0.2%). 1.64 L of refined mother liquor was collected (cytosine concentration in the refined mother liquor was 1.26 g / L). S4. Reverse Osmosis Deep Treatment: Multiple batches of secondary mother liquor, refined mother liquor, and 80L of resin regeneration wastewater are combined (the combined wastewater has a conductivity of 1350μS / cm and a COD of 650mg / L) and sent to the reverse osmosis unit for treatment at a pressure of 0.6MPa. 75.5L of purified water is recovered (the purified water has a conductivity of 45μS / cm and a COD of 35mg / L, and can be reused for fermentation). 6.5L of concentrated solute is collected and returned to step S1 for re-adsorption by the resin.

[0022] In this example, the overall recovery rate of cytosine was 78.13%.

[0023] The above embodiments are only used to illustrate the technical solutions 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 above embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the above embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for recovering cytosine from crude cytosine mother liquor, characterized in that, Includes the following steps: S1. Resin Adsorption and Elution: The combined crude mother liquor is adsorbed at a flow rate of 1–5 BV / h through a pretreated resin chromatography column. After adsorption saturation, it is eluted with 0.2–0.6 mol / L eluent at a flow rate of 0.5–2.5 BV / h (BV is the bed volume), and the eluent is collected. The resin is one of 001×7 type cation exchange resin, SP117 type cation exchange resin, D217 type anion exchange resin, or SP217 type anion exchange resin. S2. Concentration and Crude Crystallization: The eluent is concentrated at 70-80℃ and -0.075--0.09MPa to obtain a concentrated solution with a concentration of 30-50g / L. The solution is then cooled to 0-4℃ for crystallization and allowed to grow for 4-12 hours. After filtration, the filter cake is washed with purified water at 0-4℃, accounting for 20%-50% of the volume of the crystallization solution. The wet crude product and the secondary mother liquor are collected. S3. Dissolution, decolorization, and crystallization of the refined product: Dissolve the wet crude product at a concentration of 20-50 g / L of cytosine. Add activated carbon at a mass of 5-15% of cytosine for decolorization for 30-60 minutes at a decolorization temperature of 85-95℃. After decarbonization, the decolorized solution is cooled to 0-4℃ while stirring. Crystallize for 2-8 hours. After filtration, wash the filter cake with purified water at 0-4℃, accounting for 20%-50% of the volume of the crystallization solution. Dry the wet refined product and collect the mother liquor. S4. Reverse osmosis deep treatment: The secondary mother liquor, the refined mother liquor and the wastewater generated from resin regeneration are combined and sent to the reverse osmosis unit for treatment at an operating pressure of 0.6-0.8 MPa. The purified water that can be reused for fermentation is recovered and the concentrated solute is collected. The concentrated solute is returned to step S1 for resin adsorption again.

2. The method for recovering cytosine from crude cytosine mother liquor according to claim 1, characterized in that, The eluent in step S1 is 0.3-0.5 mol / L ammonia.

3. The method according to claim 1, characterized in that, The mother liquor adsorption flow rate in step S1 is 1-3 BV / h.

4. The method for recovering cytosine from crude cytosine mother liquor according to claim 1, characterized in that, The eluent flow rate in step S1 is 0.5–1.5 BV / h.

5. The method for recovering cytosine from crude cytosine mother liquor according to claim 1, characterized in that, The filter cake in step S2 is washed with purified water at 0-4°C, accounting for 20%-30% of the volume of the crystallization liquid.

6. The method for recovering cytosine from crude cytosine mother liquor according to claim 1, characterized in that, In step S3, the wet crude product is dissolved at a cytosine concentration of 30-40 g / L, and activated carbon is added at 5-8% of the cytosine mass for decolorization.

7. The method for recovering cytosine from crude cytosine mother liquor according to claim 1, characterized in that, The filtration described in steps S2 and S3 uses centrifugal filtration.