CN105567750B - Method for producing 2-keto-D-gluconic acid by two-stage semi-continuous fermentation - Google Patents
Method for producing 2-keto-D-gluconic acid by two-stage semi-continuous fermentation Download PDFInfo
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Abstract
The invention discloses a method for producing 2-keto-D-gluconic acid by fermentation with high concentration, high strength and high conversion rate by using starch hydrolysis sugar (glucose) as a raw material through two-stage semi-continuous fermentation. The invention can effectively solve the problems in the existing 2-keto-D-gluconic acid semi-continuous fermentation process, and realizes the high-concentration and high-strength fermentation of the 2-keto-D-gluconic acid while ensuring that the conversion rate of the saccharic acid is improved.
Description
Technical Field
The invention relates to a method for producing 2-keto-D-gluconic acid by fermentation by using glucose or starch hydrolysis sugar as a substrate in a two-stage semi-continuous fermentation mode, belonging to the technical field of bioengineering application.
Background
2-keto-D-gluconic acid is a basic material in heterocyclic compound synthesis, regioselective and stereoselective chemical reactions, and is mainly used as a production raw material of D-erythorbic acid and salts thereof at present. D-erythorbic acid and sodium salt thereof are safe and efficient antioxidants with remarkable cost performance, and have been widely applied to the industrial fields of food, medicine, chemical industry and the like.
The literature reports 3 methods for producing 2-keto-D-gluconic acid, such as an enzymatic method (e.g., US 4,351,902), a chemical synthesis method (e.g., US 6,018,034), and a fermentation method (e.g., US 3,255,093). Among them, the fermentation method is the most economical, efficient and environmentally friendly production method of 2-keto-D-gluconic acid at present, and thus is widely used in domestic and foreign industrial production.
The fermentation of the 2-keto-D-gluconic acid can adopt production modes such as batch fermentation, fed-batch fermentation, semi-continuous fermentation and continuous fermentation. The consumption of manpower and material resources for batch fermentation is large, the operations of charging, sterilizing, inoculating, discharging, cleaning and the like are required for each batch of fermentation, the working procedures are complicated, the fermentation period is long, and the production intensity and the product concentration in the fermentation liquor are low; fed-batch fermentation can effectively improve the product concentration in the fermentation liquor, but the fermentation production intensity is still low; compared with batch fermentation and fed-batch fermentation, the continuous fermentation has the advantages that the production intensity is obviously improved, but the continuous fermentation is easily polluted by mixed bacteria, the equipment investment is larger, the concentration of a fermentation product is lower, and the fermentation conversion rate (also called saccharic acid conversion rate, namely the molar ratio of 2-keto-D-gluconic acid produced by fermentation to glucose consumed by fermentation is multiplied by 100%) is also reduced. Compared with the three fermentation modes, the semi-continuous fermentation is a more efficient 2-keto-D-gluconic acid production mode.
In the semi-continuous fermentation process, part of fermentation liquor is discharged and then the fresh culture medium is supplemented, so that nutrients and substrates can be supplemented, and metabolic harmful substances are diluted, so that the effects of relieving product inhibition and avoiding accumulation of metabolic byproducts are achieved, the culture environment of microorganisms is improved, the stability of thallus activity is favorably maintained, and the continuous synthesis of fermentation target products is facilitated.
The prior 2-keto-D-gluconic acid single-stage semi-continuous fermentation process (Chinese patent ZL200810107055.7) has the main problem that ① needs to ensure the quality of a fermentation product, the glucose concentration in the fermentation liquid during each discharging (or feeding) process is lower than 2.0g/L, but the catalytic activity of thalli is obviously reduced, so that the subsequent fermentation production intensity is obviously influenced and generally cannot exceed 6.0 g.L-1·h-1② high-concentration fermentation is difficult to realize, and the concentration of 2-keto-D-gluconic acid in the fermentation liquid is only 165.0 g.L-1Or else, the fermentation production intensity is further reduced.
The existing two-stage semi-continuous fermentation process of 2-keto-D-gluconic acid (published Japanese graduate monograph of Zhou extensive politics of Jiangsu university) has the main problem that ① controls the glucose concentration (20.0 g.L) in the fermentation liquor during the discharging of the first-stage semi-continuous fermentation in the two-stage semi-continuous fermentation process-1) The catalytic activity of the cells is improved to some extent, but it is difficult to achieve a more desirable state, and the fermentation productivity is not so much improved, and generally not more than 7.0 g.L-1·h-1② high-concentration fermentation was not achieved, and the concentration of 2-keto-D-gluconic acid in the fermentation broth during discharging was only 165.0 g.L-1Left and right.
In addition, the sugar-acid conversion rate of the existing semi-continuous fermentation process (whether a single-stage or two-stage semi-continuous fermentation process) is difficult to exceed 90 percent (usually about 89 percent).
The invention aims to develop a high-concentration, high-strength and high-conversion-rate 2-keto-D-gluconic acid fermentation process.
Disclosure of Invention
The invention discloses a method for producing 2-keto-D-gluconic acid by fermentation with high concentration, high strength and high conversion rate by using starch hydrolysis sugar (glucose) as a raw material through two-stage semi-continuous fermentation. The invention can effectively solve the problems in the existing 2-keto-D-gluconic acid semi-continuous fermentation process, and realizes the high-concentration and high-strength fermentation of the 2-keto-D-gluconic acid while ensuring that the conversion rate of the saccharic acid is improved.
The general technical scheme of the invention is as follows: by reducing the glucose concentration in the first fermentation medium (not more than 150.0 g.L)-1) The activity of the bacteria for catalytically converting glucose into 2-keto-D-gluconic acid is improved, and the glucose concentration is reduced to 20.0-30.0 g.L in the first-stage fermentation process at the stage with higher catalytic activity-1) Transferring part of the primary fermentation liquid into a secondary fermentation tank to serve as seeds for secondary fermentation, and taking the rest fermentation liquid as seeds for primary fermentation; besides trace nitrogen sources in starch hydrolysis sugar, no other nitrogen sources are added into the second-stage fermentation culture medium, the glucose concentration of the second-stage fermentation culture medium is improved, and the growth of thalli in the second-stage fermentation process is controlled to reduce the consumption of glucose by the physiological metabolism of the thalli. The microorganism for converting glucose into 2-keto-D-gluconic acid adopted by the invention is Pseudomonas proteus preserved in the common microorganism center of China microorganism culture preservation management committee of China academy of sciences, China, institute No. 3 of microbiology, No. 1 of West Lu, 3 of the North Cheng, Naja, Beijing, and is classified and named as Pseudomonas plecoglossicida, the preservation time is 2013, 1 month and 18 days, and the preservation number is CGMCC No. 7150.
The following is a detailed description of the technical solution of the present invention:
the following operations were performed according to the sterile procedure.
(1) Microorganisms having the ability to convert glucose to 2-keto-D-gluconic acid, such as Pseudomonas (Pseudomonas), Serratia (Serratia), Erwinia (Erwinia), Arthrobacter (Arthrobacter), Gluconobacter (Gluconobacter), Alcaligenes (Alcaligenes), Acetobacter (Acetobacter), and the like, are cultured in a fermentation vessel. The microorganism cultured in this step serves as a starting seed for the two-stage semi-continuous fermentation.
(2) Inoculating the initial seeds into a first stage fermentation tank filled with a first stage fermentation medium (the amount of the fermentation medium is fermentation broth)60-85% of the volume of the fermentation tank), and culturing at a certain temperature by aeration and stirring. The glucose concentration in the primary fermentation medium is generally not more than 150.0 g.L-1Otherwise, the fermentation productivity is reduced.
(3) When the concentration of glucose in the primary fermentation liquid is reduced to 20.0-30.0 g.L-1In the process, a certain volume of primary fermentation broth (V, about 50-90% of the total amount of the primary fermentation broth) is transferred to a secondary fermentation tank.
(4) The first stage fermentation tank is supplemented with the first stage fermentation medium to the original volume (V)1About 70% of the volume of the fermentation tank), and the second-stage fermentation medium is fed to the second-stage fermentation tank to a predetermined volume (V)2About 80% of the volume of the fermentation tank), and then separately cultured under aeration and agitation at a certain temperature. In addition to trace nitrogen sources in starch hydrolysis sugar, no additional nitrogen source is added to the secondary fermentation medium, otherwise the saccharic acid conversion rate of the fermentation is affected.
(5) When the glucose concentration in the secondary fermentation liquid is reduced to 2.0 g.L-1Thereafter, the whole was discharged from the second-stage fermentor to recover 2-keto-D-gluconic acid (salt).
(6) The emptied second fermenter receives the first fermentation broth and is fed to a defined volume (V)2) Then, the culture was performed under aeration and agitation at a certain temperature.
(7) And (4) repeating the operation processes from (3) to (6) until the fermentation shows abnormal signs.
The carbon source in the culture medium is glucose (starch hydrolysis sugar), and the nitrogen source is corn steep liquor, yeast extract, beef extract or urea. The concentration of glucose (calculated as anhydrous glucose) in the primary fermentation medium is 100-150 g.L-1Preferably, the glucose concentration is 140 g.L-1Left and right; the nitrogen content of the primary fermentation medium is 0.3-2.0 g.L-1The nitrogen content is preferably 1.0 g.L-1Left and right. The carbon-nitrogen ratio (the mass concentration of glucose in the culture medium to the nitrogen content) of the secondary fermentation medium is 750: 1-870: 1, and the glucose concentration is 260-450 g.L-1(hydrolysis of the sugar stock solution with starch) and a nitrogen content of 0.32 to 0.55 g.L-1。
The fermentation of the invention is carried out at a pH of 4.0-7.0, and the pH of the fermentation is suitably about 5.5. The pH value of the fermentation is controlled by a neutralization method, and the neutralizing agent is calcium carbonate, sodium hydroxide, calcium oxide, calcium hydroxide, sodium carbonate, ammonia gas or ammonia water and the like.
The fermentation temperature of the method is 28-41 ℃, the suitable temperature of the first stage fermentation is 30-33 ℃, and the suitable temperature of the second stage fermentation is 33-35 ℃.
The fermentation of the invention needs to be carried out under aerobic condition, and the ventilation volume is about 0.5-2.0 v.v.-1·min-1The aeration rate is suitable for ensuring that the minimum dissolved oxygen concentration in the fermentation liquor in the fermentation process is not less than 0.5 percent.
The fermentation of the invention can be carried out under the normal pressure, but the pressure of 0.01-0.03 MPa is preferably selected to avoid the pollution of the mixed bacteria to the fermentation.
Drawings
FIG. 1 is a schematic diagram of two-stage semi-continuous fermentation operation
Detailed Description
Example 1
The culture of initial seeds, the first-stage semi-continuous fermentation and the second-stage semi-continuous fermentation respectively adopt 5L, 30L and 50L of automatic control mechanical stirring fermentation tanks, and the fermentation system has the functions of automatic temperature control, stepless speed regulation, measurement of the concentration of dissolved oxygen in a culture solution, pH value and the like.
Pseudomonas proteorum (Ps) CGMCC No.7150 (deposited in China general microbiological culture Collection center) was inoculated into 3.0L of a culture medium containing 20.0 g.L-1Glucose, 10.0 g.L-1Corn steep liquor, 2.0 g.L-1Urea, 2.0 g.L-1KH2PO4、0.5g·L-1MgSO4·7H2O and 2.5 g.L-1Light CaCO3(pH6.7 before sterilization), the stirring speed was controlled at 400 r.min-1Temperature 30 ℃, pot pressure 0.02MPa, and ventilation 0.5 v.v-1·min-1Culturing for 14h (OD of seed liquid)650nm＝12.4，pH＝6.95)。
The cultured initial seeds are about 10 percentThe inoculation amount is 21.0L and 140.0 g.L-1Starch hydrolysis sugar (calculated as glucose) 15.0 g.L-1Corn steep liquor and 40.0 g.L-1Light CaCO3In the first-stage fermentation culture medium, the stirring speed is controlled to be 440 r.min-1At 31 ℃, a pot pressure of 0.02MPa and a ventilation volume of 1.6 v.v-1·min-1Culturing for 10h, the glucose concentration in the primary fermentation liquid is 21.4 g.L-1，OD650nmThe concentration of 6.6, 2-keto-D-gluconic acid was 108.70 g.L-1. At this time, about 12.6L of the primary fermentation broth was transferred to the secondary fermentation tank.
Adding 19.0L of secondary fermentation medium (medium composition: 265.0 g.L of starch hydrolysis sugar) into the secondary fermentation tank-1Light CaCO375.0g·L-1) At a stirring speed of 440 r.min-1At a temperature of 33 ℃, a pot pressure of 0.02MPa and a ventilation volume of 1.6 v.v-1·min-1Fermenting for 14h under the condition of (1), and reducing the glucose concentration in the secondary fermentation liquid to 0.17 g.L-1The concentration of the 2-keto-D-gluconic acid reaches 211.81 g.L-1. Discharging and recovering 2-keto-D-gluconic acid (salt), and receiving the first-stage fermentation liquid by the emptied second-stage fermentation tank and performing a new round of second-stage fermentation according to the second-stage fermentation operation.
While the secondary fermentation operation is carried out, about 12.6L of primary fermentation medium is added into the primary fermentation tank, the fermentation is carried out for 7 hours according to the primary fermentation conditions, and the glucose concentration in the primary fermentation liquid is reduced to 20.8 g.L-1，OD650nmThe concentration of 6.8, 2-keto-D-gluconic acid was 110.54 g.L-1. At this time, about 12.6L of the primary fermentation broth was transferred to another secondary fermentation tank for secondary fermentation.
The continuous 8 rounds of primary fermentation and secondary fermentation are respectively carried out according to the method, the whole fermentation process lasts for 80h (the average fermentation time of each round is 22.25h, wherein the average time of the primary fermentation is 8.25h, the average time of the secondary fermentation is 14.00h), 55.568kg of glucose is consumed in total, 54.500kg of 2-keto-D-gluconic acid is produced, and the average mass concentration of the 2-keto-D-gluconic acid in the fermentation liquor is 215.60 g.L-1The average fermentation production intensity is 9.69 g.L-1·h-1The average conversion of sugar to acid was 91.00%.
Example 2
The fermentation equipment and the cultivation of the starting seeds are the same as in example 1.
The primary and secondary fermentations were also carried out in the same manner as in example 1, except that no CaCO was added to the fermentation medium3The pH control in the fermentation process is realized by adding 2.0mol/L NaOH solution (when the pH in the fermentation liquor is lower than 5.3, the NaOH solution is automatically controlled to be added to reach 5.3-5.5).
Performing continuous 8 rounds of primary fermentation and secondary fermentation respectively according to the above method, wherein the fermentation process lasts for 90h (the average fermentation time of each round is 24.25h, wherein the average time of the primary fermentation is 8.25h, and the average time of the secondary fermentation is 16.00h), and producing 53.970kg of 2-keto-D-gluconic acid, and the average mass concentration of the 2-keto-D-gluconic acid in the fermentation liquid is 213.49 g.L-1The average fermentation production intensity is 8.80 g.L-1·h-1The average conversion rate of saccharic acid was 90.12%.
Example 3
The fermentation equipment and the cultivation of the starting seeds are the same as in example 1.
The procedure for the primary and secondary fermentations was also the same as in example 1, except that the secondary fermentation medium consisted of: glucose (concentrated starch hydrolysis sugar) 350.0 g.L-1Light CaCO3100.0g·L-1。
The primary fermentation and the secondary fermentation are continuously carried out for 8 times respectively according to the method, the whole fermentation process lasts for 110.00h (the average fermentation time of each time is 28.25h, wherein the average time of the primary fermentation is 8.25h, the average time of the secondary fermentation is 20.00h), 68.488kg of glucose is consumed in total, 68.300kg of 2-keto-D-gluconic acid is produced, and the average mass concentration of the 2-keto-D-gluconic acid in the fermentation liquor is 270.17 g.L-1The average fermentation production intensity is 9.56 g.L-1·h-1The average conversion of sugar to acid was 92.53%.
Example 4
For comparison with the method provided by the present invention.
The initial seed was cultured in the same manner as in example 1. Single-stage semi-continuous fermentation was carried out using a 30L self-controlled fermentor. The fermentation medium comprises the following components: starch hydrolysis sugar (calculated as glucose) 162.0 g.L-115.0 g.L of corn steep liquor-1Light CaCO345.0g·L-1. Inoculating the cultured initial seed into 21.0L sterilized fermentation medium at an inoculation amount of about 10%, and controlling stirring speed to 440r min-1At 31 ℃, a pot pressure of 0.02MPa and a ventilation volume of 1.6 v.v-1·min-1Culturing for 20h, and the glucose concentration in the fermentation broth is 1.64 g.L-1，OD650nmThe concentration of 5.8, 2-keto-D-gluconic acid was 155.26 g.L-1. 12.6L of fermentation broth (2-keto-D-gluconic acid recovered) was rapidly discharged, and then supplemented with the same volume of fermentation medium, and a new round of fermentation was performed according to the fermentation method described above (about 16 h). Repeating the steps for 7 times, wherein the whole fermentation process lasts for 132 hours, the total consumed glucose is 17.690kg, the 2-keto-D-gluconic acid is produced by 16.875kg, and the average mass concentration of the 2-keto-D-gluconic acid in the fermentation liquid is 154.53 g.L-1The average fermentation production intensity is 6.09 g.L-1·h-1The average conversion of sugar acid was 88.51%.
Claims (5)
1. A process for producing 2-keto-D-gluconic acid by two-stage semi-continuous fermentation, characterized in that the glucose concentration in the first fermentation medium is reduced to not more than 150.0 g.L-1The activity of the bacteria for catalytically converting glucose into 2-keto-D-gluconic acid is improved, and the glucose concentration is reduced to 21.4-30.0 g.L in the stage with higher catalytic activity, namely the primary fermentation process-1Then transferring 50-90% of the total volume of the primary fermentation liquid into a secondary fermentation tank to serve as seeds for secondary fermentation, and taking the rest fermentation liquid as seeds for primary fermentation; adding no other nitrogen source in the second stage fermentation culture medium except the trace nitrogen source in starch hydrolysis sugar, simultaneously increasing the glucose concentration, and controlling the growth of thallus in the second stage fermentation process to reduce the physiological metabolism of thallus to grapeConsumption of sugar;
the carbon source in the culture medium is glucose, and the nitrogen source is corn steep liquor, yeast extract, beef extract or urea; the concentration of glucose (calculated as anhydrous glucose) in the primary fermentation medium is 100-150 g.L-1(ii) a The nitrogen content of the primary fermentation medium is 0.3-2.0 g.L-1(ii) a The carbon-nitrogen ratio of the secondary fermentation medium is 750: 1-870: 1, the carbon-nitrogen ratio of the secondary fermentation medium is the ratio of the mass concentration of glucose in the medium to the nitrogen content, and the glucose concentration is 260-450 g.L-1The corresponding nitrogen content is 0.32 to 0.55 g.L-1。
2. The method of claim 1, wherein the method comprises the steps of:
(1) culturing a microorganism having converted glucose into 2-keto-D-gluconic acid in a fermentation vessel, the microorganism being one of Pseudomonas (Pseudomonas), Serratia (Serratia), Erwinia (Erwinia), Arthrobacter (Arthrobacter), Gluconobacter (Gluconobacter), Alcaligenes (Alcaligenes), or Acetobacter (Acetobacter), the microorganism cultured in this step serving as a starting seed for two-stage semi-continuous fermentation;
(2) inoculating the initial seeds into a first-stage fermentation tank filled with a first-stage fermentation medium according to a certain proportion, wherein the filling amount of the fermentation medium is 60-85% of the volume of the fermentation tank, and ventilating, stirring and culturing at a certain temperature; the glucose concentration in the first-stage fermentation medium is not more than 150.0 g.L-1；
(3) When the concentration of glucose in the primary fermentation liquid is reduced to 21.4-30.0 g.L-1Then, transferring 50-90% of the total volume of the primary fermentation liquid into a secondary fermentation tank;
(4) supplementing a primary fermentation medium to the primary fermentation tank to an original volume V1 which is 70% of the volume of the fermentation tank, supplementing a secondary fermentation medium to a specified volume V2 which is 80% of the volume of the fermentation tank to the secondary fermentation tank, and then respectively ventilating, stirring and culturing at a certain temperature; no additional nitrogen source is added to the secondary fermentation medium except for the trace nitrogen source in the starch hydrolysis sugar;
(5) when the glucose concentration in the secondary fermentation liquid is reduced to 2.0 g.L-1Discharging all the obtained product from the second-stage fermentation tank to recover 2-keto-D-gluconate;
(6) the empty second stage fermenter receives the first stage fermentation broth and is fed to a specified volume V2, and then is cultured with aeration and agitation at a certain temperature;
(7) and (4) repeating the operation processes from (3) to (6) until the fermentation shows abnormal signs.
3. The method according to claim 2, wherein the fermentation is carried out at a pH of 4.0 to 7.0; the pH value of the fermentation is controlled by a neutralization method, and the neutralizer is one or more of calcium carbonate, sodium hydroxide, calcium oxide, calcium hydroxide, sodium carbonate, ammonia gas or ammonia water.
4. The method according to claim 2, wherein the suitable temperature for the first stage fermentation is 30 to 33 ℃ and the suitable temperature for the second stage fermentation is 33 to 35 ℃.
5. The method of claim 2, wherein the fermentation is carried out under aerobic conditions and the aeration rate is 0.5-2.0 v.v.-1·min-1Ensuring that the lowest dissolved oxygen concentration in the fermentation liquor is not less than 0.5 percent of ventilation capacity in the fermentation process.
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