Method for producing potash fertilizer by adjusting pH of glucuronic acid conversion liquid with KOH

Abstract

The invention provides a method for producing a potash fertilizer by adjusting the pH value of a glucuronic acid conversion solution with KOH, and relates to the technical field of fertilizers, and the method comprises the following steps: taking an inositol solution as a substrate, adding a recombinant escherichia coli thallus concentrated solution containing inositol oxidase, adjusting the pH value to 7.5-8.5 with KOH, and carrying out a conversion reaction at the temperature of 28-30 DEG C to obtain a conversion solution containing glucuronic acid; and filtering the conversion liquid, collecting a clear liquid and a concentrated liquid, carrying out resin replacement desalination and hydrochloric acid desorption on the clear liquid, and evaporating and concentrating the desorption liquid to obtain the potash fertilizer. When the potassium fertilizer is prepared, due to the protection effect of K < + > in KOH, the recombinant escherichia coli keeps good activity, the conversion efficiency of inositol is improved, the generated desalting liquid does not contain ammonium ions, and environmental pollution is avoided.

Description

Technical Field

[0001] The invention mainly relates to the technical field of fertilizers, and in particular to a method for producing potash fertilizer by adjusting the pH value of glucuronic acid conversion liquid by KOH. Background Art

[0002] Potash fertilizer is a fertilizer with potassium as the main nutrient. Its full name is potassium fertilizer. Potash fertilizer plays an important role in agricultural production and is one of the key factors in improving crop yield and quality.

[0003] In the prior art, in the industry of preparing glucuronic acid, ammonia water is used to adjust the pH of the conversion liquid, and then a cation exchange resin is used for purification, which will lead to a high ammonium ion content in the regeneration liquid, causing the ammonia nitrogen content in the waste liquid to exceed the standard, causing environmental pollution; and when whole-cell catalysis is performed, the catalytic efficiency decreases significantly after the active microorganisms catalyze once. Therefore, how to prepare glucuronic acid while ensuring that the ammonia nitrogen content in the final waste liquid does not exceed the standard, and the active microorganisms can be reused after whole-cell catalysis, is a problem that needs to be considered and solved urgently. Summary of the invention

[0004] In order to solve the technical problems in the prior art, the present invention provides a method for producing potash fertilizer by adjusting the pH of glucuronic acid conversion liquid by KOH, characterized in that it comprises the following steps:

[0005] Using inositol solution as substrate, adding recombinant Escherichia coli cell concentrate containing inositol oxidase, adjusting pH to 7.5-8.5 with KOH, carrying out conversion reaction at a temperature of 28-30° C., and obtaining conversion solution containing glucuronic acid;

[0006] The conversion liquid is filtered, and the clear liquid and the filtered concentrated liquid are collected. The clear liquid is subjected to resin replacement desalination and hydrochloric acid analysis, and the analyzed liquid is evaporated and concentrated to obtain potash fertilizer.

[0007] Compared with the prior art, the present invention uses KOH instead of ammonia water in the prior art to adjust the pH of the conversion reaction between the recombinant Escherichia coli containing inositol oxidase and the inositol substrate when preparing potash fertilizer. Firstly, due to the K + The protective effect of the invention makes the recombinant Escherichia coli containing inositol oxidase maintain good activity when the recombinant Escherichia coli contains inositol oxidase and inositol undergoes a conversion reaction, thereby improving the conversion efficiency of inositol. Secondly, the pH of the conversion solution is adjusted by avoiding the use of ammonia water, so that the desalted solution generated by the conversion solution in the subsequent resin displacement desalination process does not contain ammonium ions, thereby avoiding environmental pollution; finally, after the conversion solution is subjected to resin displacement desalination and hydrochloric acid analysis, the analytical solution is evaporated and concentrated to obtain potash fertilizer. The preparation method of the invention provides a new idea for the production and preparation of potash fertilizer.

[0008] Further, the filtered concentrate is recycled for conversion reaction of the inositol solution.

[0009] Compared with the prior art, the technical solution of the present invention uses KOH solution to adjust the pH value during the conversion process. + The protective effect of the enzyme makes it possible for the recombinant Escherichia coli in the filtered concentrate obtained after the transformation to maintain good biological activity and to be recycled for the transformation of the inositol solution, thereby allowing the recombinant Escherichia coli containing inositol oxidase to be reused, thereby improving its utilization rate and reducing the fermentation cost.

[0010] Furthermore, the OD of the recombinant E. coli concentrate containing inositol oxidase was 600 The value is 60-80, and the recombinant Escherichia coli cell concentrate containing inositol oxidase accounts for 10-20% of the volume of the inositol solution.

[0011] Furthermore, the preparation method of the recombinant Escherichia coli cell concentrate containing inositol oxidase is as follows: streak the inositol oxidase recombinant Escherichia coli on LB plate medium, inoculate the activated monoclonal colony in LB liquid medium, and culture the bacterial solution until the OD value reaches 0. 600 The value is 1.6-1.8, and bacterial solution is obtained;

[0012] Inoculate the bacterial solution into the seed tank and the seed culture medium at a rate of 0.5-2% of the weight of the seed culture medium. Cultivate until the bacterial solution OD 600 The value is 3 to 4, and the seed solution is obtained;

[0013] The seed liquid was inoculated into the fermentation tank at an inoculation amount of 5-10% of the weight of the fermentation medium. The pH was adjusted with 20wt% potassium hydroxide. During the fermentation process, the feed liquid was supplemented to maintain the DO value at 20-30%. The culture was cultured in the fermentation medium until the bacterial liquid OD 600 The value is 60-80, and the inducer is added to induce at a constant temperature for 10-14 hours. The wet cells are collected by centrifugation, and the wet cells are resuspended to obtain a concentrated solution, which is the recombinant Escherichia coli cell concentrated solution containing inositol oxidase.

[0014] Furthermore, the composition of each liter of seed culture medium is as follows: yeast powder 4-6g / L, peptone 9-11g / L, sodium chloride 9-11g / L, and the balance is water; the composition of each liter of fermentation culture medium is as follows: citric acid 2-4g / L, potassium dihydrogen phosphate 14-20g / L, dipotassium hydrogen phosphate 5-10g / L, potassium sulfate 4-10g / L, glycerol 60-80g / L, magnesium sulfate 0.6-2.0g / L, and the balance is water; the composition of the feed culture medium used is as follows: glycerol 500-600g / L, magnesium sulfate 2-3g / L, and the balance is water; the composition of the inducer used is as follows: L-arabinose 2.0-3.0g / L, and the balance is water.

[0015] Furthermore, the concentration of the inositol solution is 50-70 g / L.

[0016] Furthermore, the conversion liquid is filtered by ceramic membrane filtration, centrifuge separation, or plate and frame filter separation.

[0017] Furthermore, the resin is a cation exchange resin, and the cation exchange resin is a macroporous styrene resin, a macroporous acrylate, or a macroporous phenolic resin.

[0018] Furthermore, the concentration of hydrochloric acid is 3-5wt%.

[0019] Furthermore, when the clear liquid is subjected to resin displacement desalination, the loading rate of the clear liquid is 1-3 BV / h, and the loading amount is 2-4 BV of the resin volume.

[0020] Further, the pH of potash fertilizer is 6.5-7.5. DETAILED DESCRIPTION

[0021] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0022] The embodiment of the present invention provides a method for producing potash fertilizer by adjusting the pH of glucuronic acid conversion liquid by KOH, characterized in that it comprises the following steps:

[0023] Using inositol solution as substrate, adding recombinant Escherichia coli cell concentrate containing inositol oxidase, adjusting pH to 7.5-8.5 with KOH, carrying out conversion reaction at a temperature of 28-30° C., and obtaining conversion solution containing glucuronic acid;

[0024] The conversion liquid is filtered, and the clear liquid and the filtered concentrated liquid are collected. The clear liquid is subjected to resin replacement desalination and hydrochloric acid analysis, and the analyzed liquid is evaporated and concentrated to obtain potash fertilizer.

[0025] In the above technical scheme, the source of the inositol solution can be directly purchased from the inositol solution product of the required concentration; solid inositol powder can also be purchased, and then the inositol solution of the required concentration can be prepared according to demand; the inositol solution can also be obtained by treating corn soaking water. It should be understood that the method of treating corn soaking water to obtain the inositol solution can refer to the prior art CN116813448A, and the specific reference can be as follows:

[0026] S1: Corn soaking water passes through chelating cationic resin at a flow rate of 2-3 BV / h;

[0027] S2: Potassium phytate adsorption: The corn soaking water treated in step S1 is passed through a weakly basic anion exchange resin at a flow rate of 2-3 BV / h, wherein the volume ratio of the chelated cationic resin to the anion exchange resin in step S1 is 1:1-2;

[0028] S3: potassium phytate analysis: the phytic acid adsorbed on the resin in step S2 is analyzed with a KOH solution, wherein the concentration of the KOH solution is 5-10% W / V and the amount used is 1.5-2.5 BV of the resin volume to obtain a potassium phytate solution;

[0029] S4: Potassium phytate concentration: The potassium phytate solution obtained in step S3 is first passed through a ceramic membrane, and then concentrated using a nanofiltration membrane to obtain a potassium phytate concentrate;

[0030] S5: Potassium phytate hydrolysis: the potassium phytate concentrate obtained in step S4 is put into a hydrolysis kettle, steam is passed through, and heated to a temperature of 150-180° C. and a pressure of 0.5-0.8 MPa, and then the temperature and pressure are maintained for 8-12 hours until the hydrolysis is completed;

[0031] S6: separation: the hydrolyzate in step S5 is cooled by flash evaporation, and then plate and frame filtration is performed. The filtrate is separated by simulated moving bed chromatography to obtain an inositol solution. The concentration of the inositol solution is treated to a suitable concentration for standby use.

[0032] In the above technical solution, the pH of the conversion reaction between the inositol solution and the recombinant Escherichia coli containing inositol oxidase can be 7.5, 8.0, or 8.5; the temperature can be 28°C, 29°C, or 30°C.

[0033] In the preparation method of the present invention, after the inositol solution and the recombinant Escherichia coli containing inositol oxidase are fully mixed, KOH is used to adjust the pH of the mixed solution under the above temperature conditions to reach the pH value conditions of the present invention. Under the above temperature and pH value range conditions, the inositol solution and the recombinant Escherichia coli containing inositol oxidase can fully undergo a conversion reaction, and due to K + The protective effect of KOH makes it possible for the recombinant E. coli containing inositol oxidase to maintain good activity when it undergoes a conversion reaction with inositol, thereby improving the conversion efficiency of inositol. +The protective effect of the invention enables the recombinant Escherichia coli in the filtered concentrated solution obtained after the conversion to maintain good biological activity, and can continue to be mixed with the inositol solution to carry out the conversion reaction within the temperature and pH parameter range of the invention. Moreover, because when adjusting the pH of the conversion solution, the KOH solution is used instead of the ammonia water used in the prior art to adjust the pH of the conversion solution, so that the desalted solution generated by the conversion solution in the subsequent resin displacement desalination process does not contain ammonium ions, thereby avoiding the problem of excessive nitrogen content in sewage, thereby avoiding environmental pollution; thereafter, the conversion solution is subjected to resin displacement desalination and hydrochloric acid analysis, the potassium salt in the conversion solution is fully adsorbed by the tree branches, and then the resin is fully analyzed with hydrochloric acid to obtain the analysis solution, and the analysis solution is evaporated and concentrated to obtain potash fertilizer. The preparation method of the invention provides a new idea for the production and preparation of potash fertilizer.

[0034] In some embodiments, the filtrate concentrate is recycled for the conversion reaction of the inositol solution.

[0035] It should be understood that the filtered concentrate can be centrifuged at 5000-8000 rpm to collect the bacterial cells, which can partially or completely replace the recombinant Escherichia coli containing inositol oxidase and react with the inositol solution, and the OD of the bacterial solution containing the bacterial cells needs to be ensured. 600 The technical solution of the embodiment of the present invention allows the recombinant Escherichia coli containing inositol oxidase to be reused, thereby improving the utilization rate of the recombinant Escherichia coli containing inositol oxidase and reducing the fermentation cost.

[0036] In some embodiments, the OD of the recombinant E. coli cell concentrate containing inositol oxidase is 600 The value is 60-80, and the recombinant Escherichia coli cell concentrate containing inositol oxidase accounts for 10-20% of the volume of the inositol solution.

[0037] Specifically, the OD of the recombinant Escherichia coli cell concentrate containing inositol oxidase was 600 The value can be 60, 70, or 80; the volume of the recombinant Escherichia coli concentrate containing inositol oxidase can be 10%, 15%, or 20% of the inositol solution.

[0038] In the preparation method of the present invention, when the OD of the recombinant Escherichia coli cell concentrate containing inositol oxidase is 600 The value and the volume ratio of the inositol solution to the inositol solution are within the above ranges, which can provide an appropriate amount of bacteria for the conversion reaction and fully react with the inositol solution to undergo a conversion reaction.

[0039] In some embodiments, the preparation method of the recombinant Escherichia coli cell concentrate containing inositol oxidase is as follows: streak the inositol oxidase recombinant Escherichia coli on LB plate medium, inoculate the activated monoclonal colony in LB liquid medium, and culture the bacterial solution until the OD value is 600 The value is 1.6-1.8, and bacterial solution is obtained;

[0040] Inoculate the bacterial solution into the seed tank and the seed culture medium at a rate of 0.5-2% of the weight of the seed culture medium. Cultivate until the bacterial solution OD 600 The value is 3 to 4, and the seed solution is obtained;

[0041] The seed liquid is inoculated into a fermentation tank, the inoculation amount is 5-10% of the weight of the fermentation medium, 20wt% potassium hydroxide is used to adjust the pH, the feed liquid is supplemented during the fermentation process, the DO value is maintained at 20-30%, the bacterial liquid is cultured in the fermentation medium until the OD600 value of the bacterial liquid is 60-80, an inducer is added to induce at a constant temperature for 10-14 hours, the wet bacterial cells are collected by centrifugation, the wet bacterial cells are resuspended, and a concentrated solution is obtained, which is a recombinant Escherichia coli bacterial cell concentrated solution containing inositol oxidase.

[0042] For example, the method for preparing the recombinant Escherichia coli cell concentrate containing inositol oxidase may be:

[0043] S1: The inositol oxidase recombinant Escherichia coli was streaked and cultured on LB plate medium at 37°C for 18 h;

[0044] S2: Inoculate the single clone selected from the plate in step S1 into LB liquid culture medium, culture at 220 rpm and 37°C until the OD600 value of the bacterial solution is 1.7;

[0045] S3: Inoculate the bacterial solution in step S2 into the seed tank, and the inoculation amount is 1% of the weight of the seed culture medium; the initial pH of the seed solution is 7.0, and the initial ventilation flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, culture at a fermentation temperature of 37°C and a rotation speed of 200rpm; during the culture process, adjust the ventilation flow rate according to the dissolved oxygen DO value, maintain the DO value at about 45%, and culture until the bacterial liquid OD 600 The value is 3.5, and the seed solution is obtained;

[0046] S4: The seed solution in step S3 is inoculated into the fermentation tank, and the inoculation amount is 7% of the weight of the fermentation medium; the initial pH value of the fermentation liquid is 7.0, and the initial ventilation flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, ferment at a temperature of 37°C, a rotation speed of 400rpm, and adjust the pH with 20wt% potassium hydroxide; supplement the feed solution in time during the fermentation process to maintain the DO value at 25%, and cultivate until the OD of the fermentation liquid600 The value was 70; the inducer L-arabinose was added, the culture was induced at 30°C for 12 hours, and the wet cells were collected by centrifugation at 3000 rpm;

[0047] S5: The wet cells obtained in step S4 were resuspended, 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate were weighed, and water was added to a total volume of 1000 mL, and stirred until they were completely dissolved to obtain phosphate buffer. 50 g of wet cells were dissolved in the buffer, and stirred to mix well. Finally, OD 600 A concentrate with a value of 70.

[0048] In some embodiments, the composition of each liter of seed culture medium is as follows: yeast powder 4-6 g / L, peptone 9-11 g / L, glycerol 2-5 g / L, and the balance is water; the composition of each liter of fermentation medium is as follows: citric acid 2-4 g / L, potassium dihydrogen phosphate 14-20 g / L, dipotassium hydrogen phosphate 5-10 g / L, potassium sulfate 4-10 g / L, glycerol 60-80 g / L, magnesium sulfate 0.6-2.0 g / L, and the balance is water; the composition of the feed solution used is as follows: glycerol 500-600 g / L, magnesium sulfate 2-3 g / L, and the balance is water; the composition of the inducer used is as follows: L-arabinose 2.0-3.0 g / L, and the balance is water.

[0049] Specifically, in each liter of seed culture medium, yeast powder can be 4g / L, 5g / L, 6g / L; peptone can be 9g / L, 10g / L, 11g / L; sodium chloride can be 9g / L, 10g / L, 11g / L; citric acid can be 2g / L, 3g / L, 4g / L in each liter of fermentation medium; potassium dihydrogen phosphate can be 14g / L, 16g / L, 20g / L; potassium hydrogen phosphate can be 5g / L, 7g / L, 10g / L; sulfur can be 10g / L, 20g / L, 30g / L, 40g / L, 50g / L, 2 ... The concentration of potassium hydroxide can be 4g / L, 7g / L, and 10g / L; the concentration of glycerol can be 60g / L, 70g / L, and 80g / L; the concentration of magnesium sulfate can be 0.6g / L, 1.0g / L, and 2.0g / L; the concentration of glycerol per liter of feed solution can be 500g / L, 550g / L, and 600g / L; the concentration of magnesium sulfate can be 2g / L, 2.5g / L, and 3g / L; the concentration of L-arabinose per liter of inducer can be 2.0g / L, 2.5g / L, and 3g / L.

[0050] In some embodiments, the concentration of the inositol solution is 50-70 g / L. Specifically, the concentration of the inositol solution may be 50 g / L, 60 g / L, or 70 g / L.

[0051] In the preparation method of the present invention, the concentrated solution of recombinant Escherichia coli containing inositol oxidase is mixed with the inositol solution of the above concentration. Within this concentration range, the inositol solution can react with the recombinant Escherichia coli containing inositol oxidase to fully transform and obtain a transformation solution.

[0052] In some embodiments, the method for filtering the conversion liquid is ceramic membrane filtration, centrifuge separation, plate and frame filter separation, preferably ceramic membrane filtration.

[0053] In some embodiments, the resin is a cation exchange resin, and the cation exchange resin is a macroporous styrene resin, a macroporous acrylate, or a macroporous phenolic resin.

[0054] Specifically, the macroporous styrene resin can be SQD-67 macroporous strongly acidic styrene-based cation exchange resin, D382 strongly acidic styrene-based cation exchange resin, D72 macroporous styrene strongly acidic cation exchange resin, or macroporous strongly acidic styrene-based cation exchange resin AXD001; the macroporous acrylate can be D152 macroporous acrylic weakly acidic cation exchange resin, D301 macroporous acrylic cation exchange resin, or D113 macroporous weakly acidic acrylic cation exchange resin.

[0055] In some embodiments, the concentration of hydrochloric acid is 3-5 wt %. Specifically, the concentration of hydrochloric acid can be 3 wt %, 4 wt %, or 5 wt %.

[0056] In some embodiments, when the clear liquid is subjected to resin displacement desalination, the loading speed of the clear liquid is 1-3 BV / h, and the loading amount is 2-4 BV of the resin volume. Specifically, the loading speed of the clear liquid can be 1 BV / h, 2 BV / h, 3 BV / h, and the loading amount can be 2 BV, 3 BV, 4 BV of the resin volume.

[0057] In some embodiments, the pH of the potash fertilizer is 6.5-7.5. Specifically, the pH of the potash fertilizer may be 6.5, 7.0, or 7.5. It should be understood that the soil pH value suitable for most crops is between 6.0 and 7.5, so the potash fertilizer prepared by the preparation method of the present invention can be directly used, or the pH value of the potash fertilizer during concentration can be adjusted according to the actual conditions of plant growth to prepare a potash fertilizer with a suitable pH.

[0058] The present invention will be specifically described below by way of examples. The content of the present invention is not limited to the following examples.

[0059] Example 1

[0060] A method for producing potash fertilizer by adjusting the pH of glucuronic acid conversion liquid by KOH, comprising the following steps:

[0061] S1: Inositol oxidase recombinant Escherichia coli was streaked and cultured on LB plate medium at 37°C for 18 h;

[0062] S2: Inoculate the single clone selected from the plate in step 1 into LB liquid culture medium and culture at 220 rpm and 37°C until the bacterial solution OD 600 The value is 1.7;

[0063] S3: Inoculate the bacterial solution in step S2 into the seed tank, and the inoculation amount is 1% of the weight of the seed culture medium; the initial pH of the seed solution is 7.0, and the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, the fermentation temperature at 37°C, and the rotation speed at 200rpm for cultivation; during the cultivation process, adjust the air flow according to the dissolved oxygen, maintain the DO value at 45%, and cultivate until the seed liquid OD 600 The value is 3.5; the components of each liter of seed culture medium are 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, and the rest is water.

[0064] S4: Inoculate the seed solution in step S3 into the fermentation tank, the inoculation amount is 7.5% of the weight of the fermentation medium; the initial pH value of the fermentation liquid is 7.0, and the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, the fermentation temperature at 37°C, and the rotation speed at 400rpm for fermentation. Use 20wt% potassium hydroxide to adjust the pH. During the fermentation process, add the feed solution in time to maintain the DO value at 25%. Cultivate until the fermentation liquid OD 600 The value was 70, and the inducer L-arabinose was added. The mixture was induced at a constant temperature of 30°C for 12 hours. The wet cells were collected by centrifugation at 3000 rpm. The wet cells were resuspended, and 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate were added with water to dissolve into 1000 mL to obtain phosphate buffer. 50 g of wet cells were dissolved in the buffer and mixed well. Finally, the OD 600 The value is 70. The specific components and contents of each liter of fermentation medium are: 3 / L citric acid, 16g / L potassium dihydrogen phosphate, 7g / L dipotassium hydrogen phosphate, 7g / L potassium sulfate, 70g / L glycerol, 1g / L magnesium sulfate, and the balance is water; the inducer is 2.5g / L L-arabinose; the specific components and contents of the feed solution are: 550g / L glycerol, 2.5g / L magnesium sulfate, and the balance is water.

[0065] S5: Use 60 g / L inositol solution as substrate and add OD 600 The concentrated liquid bacteria with a pH of 70 was adjusted to 8.0 with potassium hydroxide, and a whole-cell conversion reaction was carried out at 30°C to obtain a conversion liquid containing glucuronic acid, wherein the volume of the concentrated liquid bacteria accounted for 15% of the volume of the inositol solution;

[0066] S6: filtering the conversion solution containing glucuronic acid obtained in step S5 through a ceramic membrane to obtain a ceramic membrane concentrate and a ceramic membrane clear solution;

[0067] S7: using a cation exchange resin to perform a replacement reaction to remove salt in the ceramic membrane clear solution to obtain an ion exchange liquid;

[0068] S8: Using 4% hydrochloric acid as an eluent, the cation exchange resin is eluted to obtain a analytical solution, wherein the loading rate of the ceramic membrane clear solution is 2 BV / h, and the loading amount is 3 BV of the resin volume;

[0069] S9: Evaporate and concentrate the analytical solution to a pH value of 7 to obtain potash fertilizer, which meets the national standard requirements for potash fertilizer GBT37918-2019;

[0070] S10: The ionized liquid is ultrafiltered using an ultrafiltration membrane to remove protein to obtain an ultrafiltration clear liquid, and the ultrafiltration clear liquid is decolorized using a decolorizing resin to obtain a glucuronic acid solution. The concentration of glucuronic acid in the conversion liquid is detected by high performance liquid chromatography, and the molar amount of glucuronic acid obtained is calculated and compared with the molar amount of inositol in the inositol solution, and the conversion rate is 98.6%.

[0071] Example 2

[0072] A method for producing potash fertilizer by adjusting the pH of glucuronic acid conversion liquid by KOH, comprising the following steps:

[0073] S1: Inositol oxidase recombinant Escherichia coli was streaked and cultured on LB plate medium at 37°C for 18 h;

[0074] S2: Inoculate the single clone selected from the plate in step 1 into LB liquid culture medium and culture at 220 rpm and 37°C until the bacterial solution OD 600 The value is 1.6;

[0075] S3: Inoculate the bacterial solution in step S2 into the seed tank, and the inoculation amount is 0.5% of the weight of the seed culture medium; the initial pH of the seed solution is 7.0, and the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, the fermentation temperature at 37°C, and the rotation speed at 200rpm for cultivation; during the cultivation process, adjust the air flow according to the dissolved oxygen, maintain the DO value at 50%, and cultivate until the seed liquid OD 600 The value is 3; the composition of each liter of seed culture medium is 6g / L yeast powder, 11g / L peptone, 11g / L sodium chloride, and the rest is water.

[0076] S4: Inoculate the seed solution in step S3 into the fermentation tank, the inoculation amount is 10% of the weight of the fermentation medium; the initial pH value of the fermentation liquid is 7.0, and the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, the fermentation temperature at 37°C, and the rotation speed at 400rpm for fermentation. Use 20wt% potassium hydroxide to adjust the pH. During the fermentation process, add the feed solution in time to maintain the DO value at 20%. Cultivate until the fermentation liquid OD 600The value was 60, and the inducer L-arabinose was added. The mixture was induced at a constant temperature of 30°C for 14 hours. The wet cells were collected by centrifugation at 3000 rpm. The wet cells were resuspended, and 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate were added with water to dissolve into 1000 mL to obtain phosphate buffer. 42.86 g of wet cells were dissolved in the buffer and mixed well. Finally, the OD 600 The value is 60. The specific components and contents of each liter of fermentation medium are: citric acid 2 / L, potassium dihydrogen phosphate 14g / L, dipotassium hydrogen phosphate 5g / L, potassium sulfate 4g / L, glycerol 60g / L, magnesium sulfate 0.6g / L, and the balance is water; the inducer is 2g / L L-arabinose; the specific components and contents of the feed solution are: glycerol 600g / L, magnesium sulfate 3g / L, and the balance is water.

[0077] S5: Use 50 g / L inositol solution as substrate and add OD 600 The concentrated liquid bacteria with a pH of 60 was adjusted to 7.5 with potassium hydroxide, and a whole-cell conversion reaction was carried out at 29°C to obtain a conversion liquid containing glucuronic acid, wherein the volume of the concentrated liquid bacteria accounted for 10% of the volume of the inositol solution;

[0078] S6: filtering the conversion solution containing glucuronic acid obtained in step S5 through a ceramic membrane to obtain a ceramic membrane concentrate and a ceramic membrane clear solution;

[0079] S7: using a cation exchange resin to perform a replacement reaction to remove salt in the ceramic membrane clear solution to obtain an ion exchange liquid;

[0080] S8: Using 5% hydrochloric acid as an eluent, eluting the cation exchange resin to obtain a analytical solution, wherein the loading rate of the ceramic membrane clear solution is 1 BV / h, and the loading amount is 4 BV of the resin volume;

[0081] S9: Evaporate and concentrate the analytical solution to obtain potash fertilizer when the pH value is 6.5, which meets the national standard requirements for potash fertilizer GBT37918-2019;

[0082] S10: The ionized liquid is ultrafiltered using an ultrafiltration membrane to remove protein to obtain an ultrafiltration clear liquid, and the ultrafiltration clear liquid is decolorized using a decolorizing resin to obtain a glucuronic acid solution. The concentration of glucuronic acid in the conversion liquid is detected by high performance liquid chromatography, and the molar amount of glucuronic acid obtained is calculated and compared with the molar amount of inositol in the inositol solution, and the conversion rate is 90.3%.

[0083] Example 3

[0084] A method for producing potash fertilizer by adjusting the pH of glucuronic acid conversion liquid by KOH, comprising the following steps:

[0085] S1: Inositol oxidase recombinant Escherichia coli was streaked and cultured on LB plate medium at 37°C for 18 h;

[0086] S2: Inoculate the single clone selected from the plate in step 1 into LB liquid culture medium and culture at 220 rpm and 37°C until the bacterial solution OD 600 The value is 1.8;

[0087] S3: Inoculate the bacterial solution in step S2 into the seed tank, and the inoculation amount is 2% of the weight of the seed culture medium; the initial pH of the seed solution is 7.0, and the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, the fermentation temperature at 37°C, and the rotation speed at 200rpm for cultivation; during the cultivation process, adjust the air flow according to the dissolved oxygen, maintain the DO value at 42%, and cultivate until the seed liquid OD 600 The value is 4; the composition of each liter of seed culture medium is 4g / L yeast powder, 9g / L peptone, 9g / L sodium chloride, and the rest is water.

[0088] S4: Inoculate the seed solution in step S3 into the fermentation tank, the inoculation amount is 5% of the weight of the fermentation medium; the initial pH value of the fermentation liquid is 7.0, the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, the fermentation temperature at 37°C, and the rotation speed at 400rpm for fermentation. Use 20wt% potassium hydroxide to adjust the pH. During the fermentation process, add the feed solution in time to maintain the DO value at 30%. Cultivate until the fermentation liquid OD 600 The value was 80, and the inducer L-arabinose was added, and the mixture was induced at a constant temperature of 30°C for 10 hours. The wet cells were collected by centrifugation at 3000rpm, and the wet cells were resuspended. 5.59g of dipotassium hydrogen phosphate and 0.41g of potassium dihydrogen phosphate were added with water to dissolve into 1000mL to obtain phosphate buffer. 57.14g of wet cells were dissolved in the buffer and mixed well. Finally, the OD 600 The value is 80. The specific components and contents of each liter of fermentation medium are: citric acid 4 / L, potassium dihydrogen phosphate 20g / L, dipotassium hydrogen phosphate 10g / L, potassium sulfate 10g / L, glycerol 80g / L, magnesium sulfate 2g / L, and the balance is water; the inducer is 3g / L L-arabinose; the specific components and contents of the feed solution are: glycerol 500g / L, magnesium sulfate 2g / L, and the balance is water.

[0089] S5: Use 70 g / L inositol solution as substrate and add OD 600 The concentrated liquid bacteria with a pH of 80 was adjusted to 8.5 with potassium hydroxide, and a whole-cell conversion reaction was carried out at 28°C to obtain a conversion liquid containing glucuronic acid, wherein the volume of the concentrated liquid bacteria accounted for 20% of the volume of the inositol solution;

[0090] S6: filtering the conversion solution containing glucuronic acid obtained in step S5 through a ceramic membrane to obtain a ceramic membrane concentrate and a ceramic membrane clear solution;

[0091] S7: using a cation exchange resin to perform a replacement reaction to remove salt in the ceramic membrane clear solution to obtain an ion exchange liquid;

[0092] S8: Using 3% hydrochloric acid as an eluent, eluting the cation exchange resin to obtain a analytical solution, wherein the loading rate of the ceramic membrane clear solution is 3BV / h, and the loading amount is 2BV of the resin volume;

[0093] S9: Evaporate and concentrate the analytical solution to obtain potash fertilizer when the pH value is 7.5, which meets the national standard requirements for potash fertilizer GBT37918-2019;

[0094] S10: The ionized liquid is ultrafiltered using an ultrafiltration membrane to remove protein to obtain an ultrafiltration clear liquid, and the ultrafiltration clear liquid is decolorized using a decolorizing resin to obtain a glucuronic acid solution. The concentration of glucuronic acid in the conversion liquid is detected by high performance liquid chromatography, and the molar amount of glucuronic acid calculated is compared with the molar amount of inositol in the inositol solution to obtain a conversion rate of 89.7%.

[0095] Example 4

[0096] Potassium fertilizer was prepared according to the method of Example 1, except that the concentrated liquid bacteria reacted with the inositol solution was the ceramic membrane concentrated liquid obtained in step S6 of Example 1, the ceramic membrane concentrated liquid was centrifuged at a speed of 5000 rpm, the bacteria were collected, and the collected bacteria were resuspended in phosphate buffer until the OD of the resuspended liquid was 600 The value is 70, and then mixed with the inositol solution. The composition of the phosphate buffer in the embodiment of the present invention can refer to the composition of the phosphate buffer in step S4 of Example 1.

[0097] The prepared potash fertilizer meets the national standard requirements for potash fertilizer GBT37918-2019, and the conversion rate of the prepared glucuronic acid is 97.0%.

[0098] Comparative Example 1

[0099] A method for producing a glucuronic acid solution by adjusting the pH of a glucuronic acid conversion liquid by using ammonia water, comprising the following steps:

[0100] S1: Inositol oxidase recombinant Escherichia coli was streaked and cultured on LB plate medium at 37°C for 18 h;

[0101] S2: Inoculate the single clone selected from the plate in step 1 into LB liquid culture medium and culture at 220 rpm and 37°C until the bacterial solution OD 600 The value is 1.7;

[0102] S3: Inoculate the bacterial solution in step S2 into the seed tank, and the inoculation amount is 1% of the weight of the seed culture medium; the initial pH of the seed solution is 7.0, and the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, the fermentation temperature at 37°C, and the rotation speed at 200rpm for cultivation; during the cultivation process, adjust the air flow according to the dissolved oxygen, maintain the DO value at 45%, and cultivate until the seed liquid OD 600 The value is 3.5; the composition of each liter of seed culture medium is 5g / L yeast powder, 10g / L peptone, 10g / L sodium chloride, and the rest is water.

[0103] S4: Inoculate the seed solution in step S3 into the fermentation tank, the inoculation amount is 7.5% of the weight of the fermentation medium; the initial pH value of the fermentation liquid is 7.0, and the initial air flow rate is 0.5m 3 / L, maintain the pressure at 0.02Mpa, the fermentation temperature at 37°C, and the rotation speed at 400rpm for fermentation. Use 20wt% potassium hydroxide to adjust the pH. During the fermentation process, add the feed solution in time to maintain the DO value at 25%. Cultivate until the fermentation liquid OD 600 The value was 70, and the inducer L-arabinose was added. The mixture was induced at a constant temperature of 30°C for 12 hours. The wet cells were collected by centrifugation at 3000 rpm. The wet cells were resuspended, and 5.59 g of dipotassium hydrogen phosphate and 0.41 g of potassium dihydrogen phosphate were added with water to dissolve into 1000 mL to obtain phosphate buffer. 50 g of wet cells were dissolved in the buffer and mixed well. Finally, the OD 600 The value is 70. The specific components and contents of each liter of fermentation medium are: 3 / L citric acid, 16g / L potassium dihydrogen phosphate, 7g / L dipotassium hydrogen phosphate, 7g / L potassium sulfate, 70g / L glycerol, 1g / L magnesium sulfate, and the balance is water; the inducer is 2.5g / L L-arabinose; the specific components and contents of the feed solution are: 550g / L glycerol, 2.5g / L magnesium sulfate, and the balance is water.

[0104] S5: Use 60 g / L inositol solution as substrate and add OD 600 The concentrated liquid bacteria with a pH of 70 was adjusted to 8.0 with ammonia water, and a whole-cell conversion reaction was carried out at 30°C to obtain a conversion liquid containing glucuronic acid, wherein the volume of the concentrated liquid bacteria accounted for 15% of the volume of the inositol solution;

[0105] S6: filtering the conversion solution containing glucuronic acid obtained in step S5 through a ceramic membrane to obtain a ceramic membrane concentrate and a ceramic membrane clear solution;

[0106] S7: The ceramic membrane clear liquid is ultrafiltered using an ultrafiltration membrane to remove protein to obtain an ultrafiltration clear liquid, and the ultrafiltration clear liquid is decolorized using a decolorizing resin to obtain a glucuronic acid solution. The concentration of glucuronic acid in the conversion liquid is detected by high performance liquid chromatography, and the molar amount of glucuronic acid obtained is calculated and compared with the molar amount of inositol in the inositol solution, and the conversion rate is 91.5%.

[0107] Comparative Example 2

[0108] The glucuronic acid solution was prepared according to the method of Example 4, except that the concentrated liquid bacterial cells reacted with the inositol solution used was the ceramic membrane concentrated liquid obtained in step S6 of Comparative Example 1, and the ceramic membrane concentrated liquid was centrifuged at a speed of 5000 rpm to collect the bacterial cells, and the collected bacterial cells were resuspended in a phosphate buffer until the OD of the resuspended liquid was 600 The value is 70, and then mixed with inositol solution, wherein the composition of phosphate buffer in comparative example 2 can refer to the composition of phosphate buffer in step S4 of example 1. The conversion rate of glucuraldehyde finally prepared is 52.6%.

[0109] By comparing Example 1 with Comparative Example 1, it can be seen that KOH is used to adjust the pH of the mixed solution of inositol solution and recombinant Escherichia coli containing inositol oxidase, and because K + The protective effect of K can maintain good activity of the bacteria and improve the conversion efficiency of inositol, so that the conversion rate of glucuronic acid obtained in Example 1 is higher than that in Comparative Example 1; By comparing Example 1 with Example 4, it can be seen that the conversion rate of glucuronic acid obtained in Example 4 is equivalent to that in Example 1, which further proves that K + The protective effect on the recombinant Escherichia coli containing inositol oxidase is such that the recombinant Escherichia coli in the ceramic membrane concentrate obtained after the transformation can maintain good biological activity and be reused; by comparing Example 4 with Comparative Example 2, it can be seen that the conversion rate of glucuronic acid prepared in Comparative Example 2 is significantly lower than that in Example 4, proving that the use of ammonia water for pH adjustment causes damage to the recombinant Escherichia coli cells, so that when the ceramic membrane concentrate obtained after the transformation in Comparative Example 1 undergoes a conversion reaction with the inositol solution, the conversion rate of glucuronic acid is significantly reduced, resulting in a reduced possibility of the cells being reused.

[0110] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art who is familiar with the present technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for producing potash fertilizer by adjusting the pH of glucuronic acid conversion liquid by KOH, characterized in that: The steps include: Using inositol solution as substrate, adding recombinant Escherichia coli cell concentrate containing inositol oxidase, adjusting pH to 7.5-8.5 with KOH, carrying out conversion reaction at a temperature of 28-30° C., and obtaining conversion solution containing glucuronic acid; The conversion liquid is filtered, and the clear liquid and the filtered concentrated liquid are collected. The clear liquid is subjected to resin replacement desalination and hydrochloric acid analysis, and the analyzed liquid is evaporated and concentrated to obtain potash fertilizer.

2. The method according to claim 1, characterized in that The filtered concentrate is circulated for conversion reaction of the inositol solution; and / or, The OD value of the recombinant Escherichia coli cell concentrate containing inositol oxidase is 600 A value of 60 to 80; and / or, The recombinant Escherichia coli cell concentrate containing inositol oxidase accounts for 10-20% of the volume of the inositol solution.

3. The method according to claim 1 or 2, characterized in that: The preparation method of the recombinant Escherichia coli cell concentrate containing inositol oxidase is as follows: The inositol oxidase recombinant Escherichia coli was streaked on LB plate medium, and the activated monoclonal colony was inoculated into LB liquid medium and cultured until the bacterial liquid OD 600 The value is 1.6-1.8, and bacterial solution is obtained; The bacterial solution is inoculated into a seed tank and a seed culture medium at a rate of 0.5-2% of the weight of the seed culture medium. The bacterial solution is cultured until the OD value of the bacterial solution reaches 0.5%. 600 The value is 3 to 4, and the seed solution is obtained; The seed liquid is inoculated into the fermentation tank at an inoculation amount of 5-10% of the weight of the fermentation medium, and the pH is adjusted with 20wt% potassium hydroxide. During the fermentation process, the feed liquid is supplemented to maintain the DO value at 20-30%, and the fermentation medium is cultured until the fermentation liquid OD 600 The value is 60 to 80, and the inducer is added to induce at a constant temperature for 10 to 14 hours. The wet cells are collected by centrifugation, and the wet cells are resuspended to obtain a concentrated solution, which is the recombinant Escherichia coli cell concentrated solution containing inositol oxidase.

4. The method according to claim 3, characterized in that The seed culture medium is composed of 4-6 g / L yeast powder, 9-11 g / L peptone, 9-11 g / L sodium chloride, and the balance is water; and / or, The fermentation medium is composed of 2-4 g / L citric acid, 14-20 g / L potassium dihydrogen phosphate, 5-10 g / L dipotassium hydrogen phosphate, 4-10 g / L potassium sulfate, 60-80 g / L glycerol, 0.6-2.0 g / L magnesium sulfate, and the balance is water per liter; the feed solution is composed of 500-600 g / L glycerol, 2-3 g / L magnesium sulfate, and the balance is water; the inducer is composed of 2.0-3.0 g / L L-arabinose, and the balance is water.

5. The method according to claim 1, characterized in that: The concentration of the inositol solution is 50-70 g / L.

6. The method according to claim 1, characterized in that The conversion liquid is filtered by ceramic membrane filtration, centrifuge separation, or plate and frame filter separation.

7. The method according to claim 1, characterized in that The resin is a cation exchange resin, and the cation exchange resin is a macroporous styrene resin or a macroporous acrylate.

8. The method according to claim 1, characterized in that The concentration of the hydrochloric acid is 3-5wt%.

9. The method according to claim 1, characterized in that: When the clear liquid is subjected to resin replacement desalination, the loading speed of the clear liquid is 1-3 BV / h, and the loading amount is 2-4 BV of the resin volume.

10. The method according to claim 1, characterized in that The pH of the potash fertilizer is 6.5-7.5.