CN116426577A - Calcium hydroxide as neutralizer combined with CO 2 Method for producing succinic acid by pulse feedback feed supplement fermentation - Google Patents
Calcium hydroxide as neutralizer combined with CO 2 Method for producing succinic acid by pulse feedback feed supplement fermentation Download PDFInfo
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- 238000000855 fermentation Methods 0.000 title claims abstract description 140
- 230000004151 fermentation Effects 0.000 title claims abstract description 139
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 title claims abstract description 114
- 239000001384 succinic acid Substances 0.000 title claims abstract description 54
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 title claims abstract description 36
- 239000000920 calcium hydroxide Substances 0.000 title claims abstract description 29
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- 238000000034 method Methods 0.000 claims abstract description 36
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- 230000003213 activating effect Effects 0.000 claims abstract description 6
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- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
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- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
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- KHPXUQMNIQBQEV-UHFFFAOYSA-N oxaloacetic acid Chemical compound OC(=O)CC(=O)C(O)=O KHPXUQMNIQBQEV-UHFFFAOYSA-N 0.000 description 1
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/44—Polycarboxylic acids
- C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
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Abstract
The invention provides a method for combining calcium hydroxide as a neutralizer with CO 2 A method for producing succinic acid by pulse feedback feed fermentation, the method comprising: activating a fermentation strain WS100, and then sequentially carrying out primary seed culture and secondary seed culture to obtain secondary seed liquid; and (3) culturing the secondary seed liquid in a fermentation tank to produce succinic acid, wherein the fermentation tank culturing stage comprises the following operations: monitoring the pH value of the fermentation liquor at the initial stage of the culture of the fermentation tank, and when the pH value of the fermentation liquor naturally drops to 5-6.5, adding 22-24% calcium hydroxide solution as a neutralizer until the pH value rises to 7.5-9, and stopping adding; introducing carbon dioxide gas until the pH value is reduced to 6.5-7, and stopping to naturally ferment the fermentation liquor; when succinic acid in the fermentation liquid is continuously accumulatedWhen the product naturally reduces the pH value to 5-6.5, repeating the above operation.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for combining calcium hydroxide as a neutralizer with CO 2 A method for producing succinic acid by pulse feedback feed supplement fermentation.
Background
Succinic acid is used as a synthesis precursor of chemical intermediates and is widely applied to the fields of foods, chemical industry, medicines, degradable biological materials and the like. In recent years, the technology for producing succinic acid by biological fermentation is gradually mature and applied to industrialization through continuous improvement of strains for producing succinic acid by biological fermentation (Chinese patent CN 104178443A;CN 102827800A), but the technology still has some factors based on cost consideration, such as the selection of neutralizing agents and the separation and purification of products, and prevents the application process of synthesizing succinic acid by a biological method.
Several neutralizing agents commonly used in organic acid fermentation at present are also widely used in succinic acid fermentation production. In contrast, magnesium carbonate-based neutralizers are generally considered to be the most effective in improving the fermentation yield and production strength of succinic acid (metabolic engineering of E.coli to succinic acid synthesis and tank scale-up processes, microbiology report, 2018,45 (12): 2541-2551; optimization and scale-up of fermentation conditions for E.coli FMME-N-26 to succinic acid production, process engineering report, 2022,22 (7): 854-862). The neutralizing agent based on magnesium carbonate is used as an alkaline reagent, so that succinic acid generated in the process of alkaline neutralization fermentation is utilized to avoid poisoning of acid on fermenting organisms, and magnesium ions are auxiliary factors of phosphoenolpyruvate carboxylase so as to promote accumulation of succinic acid, and CO generated in the process of neutralization 2 The method can be immobilized by the fermentation intermediate pyruvic acid/phosphoenolpyruvic acid, promotes the flow direction of pyruvic acid/phosphoenolpyruvic acid to the succinic acid synthesis path, and improves the yield of succinic acid. The disadvantages of magnesium ion neutralizers are also apparent, and the current price of magnesium carbonate is high, which is difficult to be dominant compared with the prices of sodium carbonate, sodium bicarbonate, sodium hydroxide, calcium carbonate, calcium bicarbonate, calcium hydroxide and the like. In addition, free magnesium ions are pairedThe subsequent product separation and purification process also presents challenges, as divalent cations, and presents a heavy load on the subsequent ion exchange or electrodialysis techniques, thus keeping the cost of separation high.
As a traditional organic acid fermentation neutralizer, the calcium hydroxide has the characteristics of low cost and mature matched process. Under the condition of adding sulfuric acid, the generated calcium succinate is easy to generate calcium sulfate precipitate to remove calcium ions in fermentation liquor, and the subsequent product separation and purification process of fermentation is simple, so that the succinic acid production cost is greatly reduced. However, when succinic acid is fermented, calcium hydroxide is used as a neutralizer, the fermentation yield and the sugar acid conversion rate are often lower than those of other neutralizers, and if calcium carbonate or calcium bicarbonate is used as the neutralizer, the precursor CO required by succinic acid fermentation can be supplemented in the fermentation process 2 But the neutralizing agent has weak alkalinity and can not quickly adjust the pH of the fermentation liquor.
Therefore, the method for producing the succinic acid by fermentation is required to be provided, so that the conversion rate of the succinic acid fermentation sugar acid can be improved, and meanwhile, the cost of fermentation and subsequent separation and purification can be obviously reduced.
Disclosure of Invention
The invention aims to provide a method for combining CO by taking calcium hydroxide as a neutralizer 2 Method for producing succinic acid by pulse feedback feed supplement fermentation, wherein calcium hydroxide is a neutralizer combined with CO 2 The pulse feedback feed supplement fermentation can improve the conversion rate of succinic acid fermentation sugar acid, and obviously reduce the cost of fermentation and subsequent separation and purification.
In order to solve the technical problems, the invention adopts the following technical scheme:
the invention provides a method for combining calcium hydroxide as a neutralizer with CO 2 A method for producing succinic acid by pulse feedback feed fermentation, the method comprising:
activating a fermentation strain WS100, and then sequentially carrying out primary seed culture and secondary seed culture to obtain secondary seed liquid;
and (3) culturing the secondary seed liquid in a fermentation tank to produce succinic acid, wherein the fermentation tank culturing stage comprises the following operations:
monitoring the pH value of the fermentation liquor at the initial stage of the culture of the fermentation tank, and when the pH value of the fermentation liquor naturally drops to 5-6.5, adding 22-24% calcium hydroxide solution as a neutralizer until the pH value rises to 7.5-9, and stopping adding;
introducing carbon dioxide gas until the pH value is reduced to 6.5-7, and stopping to naturally ferment the fermentation liquor;
repeating the above operation when the pH value naturally drops to 5-6.5 due to the continuous accumulation of succinic acid in the fermentation broth.
Further, the flow rate of the carbon dioxide gas is 0.1 to 0.5vvm.
Further, the basic formula of the culture solution cultured in the fermentation tank comprises the following components in percentage by mass: 7.5-8.5% glucose, 0.2-0.4% corn steep liquor.
Further, the secondary seed liquid is inoculated into the culture liquid cultivated in the fermentation tank according to the inoculation amount of 10-15 percent.
Further, the culture solution of the fermentation tank culture contains 75-85 g/L of initial glucose, and 15-25 g/L of glucose is added when the glucose content is lower than 10g/L in the fermentation tank culture.
Further, in the culture of the fermentation tank, the fermentation temperature is 35-39 ℃ and the stirring rotation speed is 200-300 r/min.
Further, the activating comprises: culturing the fermentation strain WS100 on an LB plate for 2-3 times, wherein the culture conditions are as follows: the culture temperature is 35-39 ℃ and the culture time is 12-24 h.
Further, the conditions of the primary seed culture are as follows: shake flask culture is carried out for 10 to 12 hours at the temperature of 35 to 39 ℃ and the speed of 200 to 300r/min.
Further, the conditions of the secondary seed culture are as follows: shake flask culture is carried out for 20 to 24 hours at the temperature of between 35 and 39 ℃ and at the speed of between 200 and 300 r/min; the culture solution obtained by the primary seed culture is inoculated according to the inoculation amount of 5-10 percent.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
1. the invention supplements calcium hydroxide and CO by time-sharing alternate pulse 2 Solves the problems ofAnd the problem of low succinic acid fermentation yield when calcium hydroxide is used as a neutralizer. For example, in the case of succinic acid, constant speed is automatically fed as the neutralizing agent calcium hydroxide solution, since there is no CO 2 The succinic acid is supplied, the raw material for synthesizing oxaloacetic acid from pyruvic acid/phosphoenolpyruvic acid is lacking, and the fermentation speed is slow. If at the same time, the CO gas is continuously supplemented 2 The fermentation liquor is acidified due to the generation of carbonic acid, and meanwhile, calcium hydroxide and carbonic acid fed in the fermentation tank generate calcium carbonate sediment, so that the consumption of calcium hydroxide is greatly increased, and the generation of calcium carbonate reduces CO 2 Efficiency of conversion to succinic acid. Calcium hydroxide and CO are fed in by time-sharing alternate pulse 2 The utilization efficiency of the two is improved. The results show that, relative to the uninterrupted make-up gas CO 2 By contrast, the fermentation rate and the sugar acid conversion rate of the succinic acid are obviously improved.
2. The invention uses calcium hydroxide as neutralizer to ferment succinic acid, carbon dioxide is intermittently introduced into fermentation liquor in a gas mode to supplement succinic acid to synthesize a required carbon skeleton, and the pH value of the fermentation liquor is controlled. Compared with the method which directly uses calcium carbonate as a neutralizer and a carbon skeleton provider, the succinic acid fermentation sugar acid conversion rate is improved by 1.03 times.
3. The pH value of the fermentation liquor is controlled by alternately using the calcium hydroxide solution and the carbon dioxide, so that the pH value of the fermentation liquor can be stabilized within the range of 5.0-9.0. The conversion rate of succinic acid and sugar acid is improved by 1.82 times compared with the control of directly introducing carbon dioxide.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a fermentation progress curve of example 5.
FIG. 2 is a fermentation progress curve of comparative example 2.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control. Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.
The discovery process of the technical problem of the invention is as follows:
a large number of experiments using calcium hydroxide as a succinic acid fermentation neutralizer show that the method has obvious advantages in the aspect of subsequent separation and purification cost by using only calcium hydroxide as the neutralizer, but the fermentation yield is not high. When calcium carbonate salt is added, CO is released only when the pH value of the fermentation liquid is low 2 But is unfavorable for the growth of fermenting microorganisms when the pH of the fermentation broth is low. The addition of other carbonates can result in high separation and purification costs. By CO 2 Maintaining pressure of fermentation tank or continuously introducing CO 2 When the gas is generated, indissolvable calcium carbonate is generated in a large amount in the fermentation tank, and subsequent fermentation is difficult.
To overcome the above disadvantages, we propose a method of supplementing CO by pulse mode feedback 2 The novel method improves the sugar acid conversion rate of succinic acid fermentation when only calcium hydroxide is used as a neutralizer. Relative to the continuous CO supply in some patents 2 According to the pH condition of the fermentation broth, the method pulses CO in a feedback manner 2 Effectively avoids CO when the pH of the fermentation liquor is stably controlled within a certain range 2 Continuous feeding brings about continuous acidification of fermentation liquor, which causes the problem that calcium hydroxide has to be added in a direct current way, as well asAnd the sugar acid conversion rate and the fermentation rate of succinic acid fermentation are increased.
The following will be made in connection with examples and experimental data for a calcium hydroxide as a neutralizing agent in combination with CO 2 The method for producing succinic acid by pulse feedback feed-batch fermentation is described in detail.
Example 1
(1) Strain activation:
taking escherichia coli WS100 for producing succinic acid as a fermentation strain, wherein the construction method of the escherichia coli WS100 comprises the following steps: the E.coli W is taken as an initial strain (the initial strain is purchased and has the preservation number of ATCC 9637), and lactic acid dehydrogenase gene (ldh A), alcohol dehydrogenase gene (adh E), pyruvate formate lyase gene (pflB), pyruvate oxidase gene (poxB) and acetate kinase gene (ackA) are respectively knocked out by using a Red homologous recombination system, and then the E.coli WS100 is obtained by a screening method through anaerobic growth. The detailed construction method of E.coli WS100 is described in the following literature: construction of recombinant escherichia coli with high succinic acid yield and anaerobic fermentation, food and fermentation industry 2013,31 (1) 6-10.
Activating the strain WS100 on an LB plate for 2-3 times, and culturing at 35-39 ℃ for 12-24 hours;
(2) Primary seed culture: single colony on the flat plate is selected and inoculated into 50ml LB culture medium containing 20g/L glucose, and shake flask culture is carried out for 10-12 h at 35-39 ℃ and 200-300 r/min;
(3) Secondary seed culture: inoculating the first-stage seed liquid into the second-stage seed culture medium according to the inoculation amount of 5-10%, shaking flask culture is carried out for 20-24 h at 35-39 ℃ and 200-250 r/min, and at this time, the OD of the seed liquid is equal to that of the second-stage seed liquid 600 The value is more than 15. The formula of the secondary seed culture medium is as follows: 40g/L glucose, 10g/L yeast powder, 20g/L tryptone, 1.14g/L KH 2 PO 4 ，0.9g/LK 2 HPO 4 ·12H 2 O，3.0g/L(NH 4 ) 2 SO 4 ，0.5g/L MgSO 4 ·12H 2 O。
(4) Culturing in a 10 liter fermentation tank: the basic formula of the fermentation culture solution is as follows: 8% glucose, 0.3% corn steep liquor. Pressing the second seed liquid15% of inoculation amount is inoculated into the fermentation culture solution. The fermentation medium contains an initial glucose content of 80g/L and is supplemented with 20g/L glucose when the glucose content is below 10 g/L. The fermentation temperature is 38 ℃, the stirring speed is 300r/min, and 23% calcium hydroxide solution is used as a neutralizer. No neutralizing agent is added at the initial stage of the culture stage of the fermentation tank, the pH value of the fermentation liquor naturally drops to about 5.0, a peristaltic pump is started to manually feed calcium hydroxide to raise the pH value of the fermentation liquor to about 9.0, then the addition of the neutralizing agent is stopped immediately, and CO is introduced 2 The CO is stopped when the pH value of the gas (the flow rate is 0.2 vvm) is reduced to 7.0 2 Fermenting the fermentation liquid naturally, and repeating the above operation when the pH value is naturally reduced to about 5.0 due to the continuous accumulation of succinic acid in the fermentation liquid.
(5) Detection and technology of relevant parameters in the fermentation process:
(1) measurement of biomass: 1mL of fermentation broth is taken, 1mL of 6mol/L hydrochloric acid is used for neutralizing calcium hydroxide in the fermentation broth, the fermentation broth is diluted by 2-30 times by pure water, the OD value of the diluted fermentation broth is measured by using a spectrophotometer, and the detection wavelength is 600nm.
(2) Measurement of glucose: 1mL of fermentation broth is taken, 8000r/min is centrifuged for 1min, supernatant is taken, and after dilution by a proper factor, the measurement is carried out by using a biosensor SBA 40D.
(3) Measurement of succinic acid: treating the fermentation broth with 6% sulfuric acid solution at a volume ratio of 1:1, standing for several minutes, centrifuging at 10000r/min for 10min, diluting according to growth state at a certain ratio, and measuring succinic acid content by using a high performance liquid chromatograph. The measurement conditions of the high performance liquid chromatography are as follows: bio-Rad HPX 87H; mobile phase: 5mmol/L H 2 SO 4 Solution (membrane suction filtration of 0.22 μm before use, degassing by ultrasound for 30 min); flow rate: 0.5mL/min; column temperature: 45 ℃; a detector: a PDA; detection wavelength: 210nm; sample injection amount: 10 mu L.
Wherein the sugar acid conversion (%) is calculated according to formula (1):
wherein rho and succinic acid concentration are in g/L; v, the total volume of the fermentation liquor is L; m, total sugar consumption in g. After fermentation, the concentration of the succinic acid is 52.76g/L, and the conversion rate is 69.55%.
Examples 2 to 11
Examples 2 to 11 employ fermentation conditions and experimental methods similar to those of example 1. The difference is that when the pH value of the fermentation liquor naturally drops to a certain pH value with slightly acidic property (such as pH value 5,5.5,6.0,6.5), calcium hydroxide is added to raise the pH value to an alkaline pH value (such as pH value 7.5,8.0,8.5,9.0), then the addition of the neutralizing agent is stopped immediately, and CO is introduced 2 The gas (flow rates of 0.1,0.2,0.3,0.4 and 0.5 vvm) is stopped when the pH value is reduced to a neutral pH value (such as pH value of 6,5 and 7) 2 The fermentation broth is naturally fermented, when the pH value is naturally reduced to a specific acidic pH value (for example, the pH value is 5.0,5.5,6.0,6.5) by continuously accumulating succinic acid in the fermentation broth, calcium hydroxide is added again, and the above operation is repeated, wherein the specific parameters are shown in Table 1.
Comparative example 1
The same strains, fermentation conditions and experimental methods as in example 1 were used. The difference is that CO is not introduced in the whole fermentation stage 2 And (3) taking 8% of calcium carbonate dry powder as a neutralizer. The results are shown in Table 2.
Comparative example 2
The same strains, fermentation conditions and experimental methods as in example 1 were used. The difference is that CO is not introduced in the whole fermentation stage 2 The gas and fermentation results are shown in Table 2, and the fermentation progress curve is shown in FIG. 2.
Comparative example 3
The same strains, fermentation conditions and experimental methods as in example 1 were used. The difference is that CO is introduced at constant speed in the whole fermentation stage 2 The gas (flow rate is 0.5 vvm), the fermentation result is shown in Table 2, and the fermentation process curve is shown in FIG. 2.
Experimental example 1, results measurement
The succinic acid yield and sugar acid conversion in each example and each comparative example were counted as shown in tables 1 and 2.
Table 1 experimental results of example 1, example 11
As can be seen from Table 1, the pH control mode of example 5 is preferred, the succinic acid yield reaches 85.65g/L, the sugar acid conversion rate reaches 96.75%, the fermentation process curve is shown in FIG. 1, and the pH value of the fermentation liquor can be stabilized within the range of 5.5-7.5 by alternately using the calcium hydroxide solution and the carbon dioxide to control the pH value of the fermentation liquor.
TABLE 2 comparison of fermentation results for example 5 and comparative example 3
In comparative example 1, the succinic acid content of the fermentation broth was 37.13g/L, the sugar acid conversion rate was 47.62%, and the corresponding data were lower compared with all examples of the present invention. Example 5 shows the best fermentation results, and the sugar acid conversion is increased by a factor of 1.03 in example 5 compared to comparative example 1.
In comparative example 2, the succinic acid content of the fermentation broth was 15.11g/L, the sugar acid conversion was 24.84%, and the corresponding data was lower compared to all examples, wherein the sugar acid conversion was increased by 2.89 times compared to comparative example 2 in example 5.
Compared with comparative example 3, the sugar acid conversion of succinic acid fermentation of example 5 was compared with the continuous supplementation of gas CO 2 Is improved by 1.82 times.
In conclusion, the invention overcomes the current situation that the existing strain has low succinic acid production efficiency by fermentation by using calcium hydroxide as a neutralizer, and provides a method for combining the calcium hydroxide as the neutralizer with CO 2 The method for producing the succinic acid by pulse feedback feed supplement fermentation provides a new idea for simplifying the process and synthesizing the succinic acid with low cost.
The invention provides a general pH control method for reference of fumaric acid and malic acid fermentation. Many organic acids ferment with CO 2 But CO 2 Is often in conflict with the addition of alkali such as calcium hydroxide, and according to the pH measurement result of fermentation liquor, the fermentation liquor is prepared by the alkali and CO 2 The pulse supplement is circulated in different time periods to effectively control the pH value of the fermentation liquor and improve the CO 2 The utilization rate, thereby improving the sugar acid conversion rate of the related organic acid fermentation provides a beneficial thought.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (9)
1. Calcium hydroxide as neutralizer combined with CO 2 A method for producing succinic acid by pulse feedback feed fermentation, which is characterized by comprising the following steps:
activating a fermentation strain WS100, and then sequentially carrying out primary seed culture and secondary seed culture to obtain secondary seed liquid;
and (3) culturing the secondary seed liquid in a fermentation tank to produce succinic acid, wherein the fermentation tank culturing stage comprises the following operations:
monitoring the pH value of the fermentation liquor at the initial stage of the culture of the fermentation tank, and when the pH value of the fermentation liquor naturally drops to 5-6.5, adding 22-24% calcium hydroxide solution as a neutralizer until the pH value rises to 7.5-9, and stopping adding;
introducing carbon dioxide gas until the pH value is reduced to 6.5-7, and stopping to naturally ferment the fermentation liquor;
repeating the above operation when the pH value naturally drops to 5-6.5 due to the continuous accumulation of succinic acid in the fermentation broth.
2. The method according to claim 1, wherein the flow rate of the carbon dioxide gas is 0.1 to 0.5vvm.
3. The method according to claim 1, wherein the basic formula of the culture fluid cultured in the fermentation tank comprises the following components in percentage by mass: 7.5-8.5% glucose, 0.2-0.4% corn steep liquor.
4. The method according to claim 1, wherein the secondary seed liquid is inoculated into the culture liquid cultivated in the fermenter in an inoculum size of 10 to 15%.
5. The method according to claim 1, wherein the culture broth of the fermenter culture contains an initial glucose content of 75 to 85g/L, and glucose of 15 to 25g/L is added when the glucose content is lower than 10 g/L.
6. The method according to claim 1, wherein the fermentation temperature is 35-39 ℃ and the stirring speed is 200-300 r/min in the fermentation tank culture.
7. The method of claim 1, wherein the activating comprises: culturing the fermentation strain WS100 on an LB plate for 2-3 times, wherein the culture conditions are as follows: the culture temperature is 35-39 ℃ and the culture time is 12-24 h.
8. The method of claim 1, wherein the primary seed culture conditions are: shake flask culture is carried out for 10 to 12 hours at the temperature of 35 to 39 ℃ and the speed of 200 to 300r/min.
9. The method of claim 1, wherein the secondary seed culture conditions are: shake flask culture is carried out for 20 to 24 hours at the temperature of between 35 and 39 ℃ and at the speed of between 200 and 300 r/min; the culture solution obtained by the primary seed culture is inoculated according to the inoculation amount of 5-10 percent.
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