CN114921502A - Method for producing glutaric acid by performing feedback regulation and control on nitrogen source feeding based on microbial physiological parameters - Google Patents
Method for producing glutaric acid by performing feedback regulation and control on nitrogen source feeding based on microbial physiological parameters Download PDFInfo
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Abstract
The invention relates to a method for producing glutaric acid by feedback regulation and control of nitrogen source feeding based on microbial physiological parameters, which comprises the steps of inoculating strain culture solution into a fermentation tank containing a fermentation medium, and performing feedback regulation and control of nitrogen source feeding speed according to real-time microbial physiological parameters such as oxygen consumption rate (OUR) and carbon dioxide generation rate (CER) in the fermentation process to obtain glutaric acid by fermentation. The method has the advantages of high acid production, high conversion rate and easy large-scale amplification, and lays a foundation for industrialization of the biological glutaric acid.
Description
Technical Field
The invention belongs to the field of biology, and particularly relates to a method for producing glutaric acid by controlling nitrogen source feeding based on microbial physiological parameter feedback.
Background
Glutaric acid can be used for synthesizing glutaric anhydride, and can be widely applied to the fields of rubber, medicine and the like. Furthermore, the polymerization of glutaric acid with different diamines gives novel polyamides. At present, most of glutaric acid on the market is obtained by separating adipic acid as a by-product or by a chemical reaction. The chemical synthesis of glutaric acid requires high temperature, high pressure and expensive catalyst, such as the synthesis of glutaric acid by ring opening of butyrolactone with potassium cyanide, which is a highly toxic compound, followed by hydrolysis, and the process has the disadvantages of high toxicity, high cost, low process safety and environmental friendliness. Obtaining high purity glutaric acid by separating adipic acid by-products requires multi-step crystallization, low yield, high cost, and limits further applications of glutaric acid. In recent years, the production of glutaric acid by metabolic engineering of microorganisms has been a focus of research, and microorganisms including Escherichia coli and Corynebacterium glutamicum have been widely developed to synthesize glutaric acid. The 5-aminopentanoate pathway (AMV) pathway is considered to be the most promising glutaric acid biosynthesis pathway. As shown in FIG. 1, the AMV pathway is obtained by selecting a suitable host, such as Escherichia coli or Corynebacterium glutamicum, in which lysine 2-monooxygenase (DavB) and delta-aminopentanamide (DavA) derived from Pseudomonas putida (Pseudomonas putida) are overexpressed, metabolizing lysine to 5-aminopentanoate, and further obtaining glutaric acid by 5-aminopentanoate aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). To date, studies on the biosynthesis of glutaric acid have focused mainly on the metabolic engineering of microorganisms, while few studies have been made on the fermentation process of glutaric acid. The microbial production of L-lysine requires large amounts of nitrogen sources such as ammonium sulfate and liquid ammonia, while the production of glutaric acid based on the catabolism of L-lysine requires stepwise deamination and transamination reactions. Therefore, nitrogen source feeding strategy will interfere with L-lysine and glutaric acid synthesis, however, no relevant literature reports how nitrogen source affects glutaric acid synthesis. As also shown in FIG. 1, L-lysine to 5-aminopentanamide requires oxygen and produces carbon dioxide, which is related to real-time microbial Oxygen Uptake Rate (OUR), carbon dioxide release rate (CER) physiological parameters. In the invention, a nitrogen source feeding strategy for carrying out feedback regulation based on real-time microbial physiological parameters is developed and applied to biological production of glutaric acid. The process has high acid production and high saccharic acid conversion rate, and is easy to be enlarged to an industrial scale.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for producing glutaric acid by performing feedback regulation and control on nitrogen source feeding based on microbial physiological parameters.
The invention relates to a method for producing glutaric acid by performing feedback regulation and control on nitrogen source feeding based on microbial physiological parameters, which comprises the following steps:
inoculating a strain into a fermentation tank containing a fermentation medium, and performing fermentation by using real-time microbial physiological parameters as the basis to feedback control nitrogen source feeding; wherein the microorganism physiological parameters comprise oxygen consumption rate (OUR) and carbon dioxide generation rate (CER).
Further, the culture solution of the glutaric acid producing strain is inoculated into a fermentation tank containing a fermentation culture medium, and the microorganism grows by using the culture medium under the proper culture medium and culture conditions, when the real-time OUR and CER gradually rise to 140-180m mol L -1 h -1 The microorganism enters the glutaric acid rapid synthesis stage, at which time the nitrogen source feed is started.
The nitrogen source feeding regulation is the nitrogen source feeding regulation speed, and the nitrogen source feeding regulation speed can be automatically fed back and regulated based on real-time OUR and CER, and can also be manually regulated according to real-time OUR and CER.
Further, when the OUR and CER in the acid production period are lower than 140- -1 h -1 Increasing the nitrogen source flow acceleration rate to maintain OUR and CER at 140- -1 h -1 More preferably 160-180 m mol L -1 h -1 。
The nitrogen source is one or more of ammonia water, liquid ammonia, ammonium sulfate solution, ammonium chloride solution, urea solution, ammonium carbonate and ammonium bicarbonate solution, preferably ammonium sulfate solution, wherein the solution is aqueous solution.
When ammonia is used for nitrogen source flow, the pH of the fermentation broth can be controlled with ammonia at the same time to maintain a pH of 6.7-7.2, or with sodium hydroxide solution, the concentration of ammonia is typically 25-28% (w/v) and the concentration of sodium hydroxide solution is typically 5-40% (w/v).
Further preferably, a nitrogen source is provided by feeding 10-50% (w/v) ammonium sulfate solution, and the pH value of the fermentation broth is regulated to 6.7-7.2 by 5-40% (w/v) sodium hydroxide solution.
The fermentation process also comprises feeding glucose solution to make the concentration of glucose in the fermentation liquor be 0.5-1% (w/v). And feeding a glucose solution, wherein the concentration of the glucose solution is 50-70% (w/v).
The fermentation process parameters comprise: the inoculation amount is 10-20%, the fermentation temperature is 35-37 ℃, the pH value of the fermentation liquor is controlled to be 6.7-7.2, the stirring speed is related to Dissolved Oxygen (DO), the DO is maintained at 20-40%, the tank pressure is maintained at 0.05-0.1 MPa, and the fermentation period is 50-70 h.
The culture medium adopted by the fermentation is as follows: 3-8 g/L potassium dihydrogen phosphate, 1-3 g/L magnesium sulfate, 0.01-0.03 g/L ferrous sulfate, 0.01-0.04 g/L manganese sulfate, 5-15 g/L ammonium sulfate, 20-40 g/L glucose, 5-30 g/L corn steep liquor, 0.5-3 g/L betaine, 0.002-0.006 g/L vitamin B1, and antibiotics added into the culture medium, wherein the antibiotics are one or more of 30-200 mu g/mL ampicillin, 30-100 mu g/mL chloramphenicol and 30-200 mu g/mL kanamycin.
The strain is escherichia coli or corynebacterium glutamicum modified by metabolic engineering.
Note: the mass volume% (w/v) in the present invention is g/100 ml.
Furthermore, the glutaric acid producing strain adopted in the experiment is a metabolic engineering strain. Related researches on glutaric acid metabolism engineering bacteria are reported more. The engineering of a Glutaric acid-producing bacterium based on the AMV pathway is mainly carried out by transferring a plasmid containing a lysine 2-monooxygenase (DavB) encoding gene and a delta-aminopentanamide (DavA) encoding gene from Pseudomonas putida into a host bacterium, and further overexpressing 5-aminopentanoate aminotransferase (GabT) and succinate semialdehyde dehydrogenase (GabD, to obtain Glutaric acid, lysine is a precursor for synthesizing Glutaric acid, and thus it is also a common means to enhance the Metabolic flux of lysine. at present, Glutaric acid-producing bacteria are mostly engineered Escherichia coli or Corynebacterium glutamicum. for example, studies by Han et al report that glutamic acid production by systems metabolism engineering of L-lysine-overproducing bacterium (2020, Pn117, 30328-30334), studies by m et al report that the activity of nucleic acid of Escherichia coli, a C5 dicarboxylic acid platform for chemical (2019, Metab. Eng.51, 99-109), research report by Li et al Targeting metabolic drying and interaction in L-lysine catalyst for high-level reaction process (2019, Nat. Commun.10,3337), research report by Rohles et al System metabolism Engineering of Corynebacterium luminescence for the reaction product of the Carbon-5platform chemicals 5-amino catalysts and butyl catalysts (2016, Microb. cell. factor 15,1-13), research report by Adkins et al Engineering for reaction product of the Carbon-5platform chemicals and reaction product (2016, Microb. cell. factor.15, 1-13), research report by Adkins et al Engineering for reaction product of the carbohydrate-5 platform chemicals and reaction product (2015. cell. filtration. admixture, 2-3, reaction report by Adkins et al reaction product for reaction product of reaction product for reaction product, 2015-biological reaction product, and reaction product of reaction product, reaction product of the microorganism, cell. 12-3, biological reaction product of the reaction product of the reaction of the.
Further, the process is exemplified by the synthesis of glutaric acid by the metabolic engineering strain Escherichia coli LQ-1 constructed in the above literature based on the AMV pathway in the laboratory of the present inventors, and the claims are not limited to the present laboratory species, the relevant medium and the culture method.
The biological method for fermenting glutaric acid comprises the processes of glycerinum pipe seed activation, shake flask seed culture, seed tank seed culture and fermentation.
After the glycerol tube is naturally thawed, taking 300uL of sterilized gun head on a plate culture medium, spreading the solution as much as possible, and carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 24h, wherein the plate culture medium is as follows: 4-10 g/L potassium dihydrogen phosphate, 0.3-1 g/L magnesium sulfate heptahydrate, 2-6 g/L ammonium sulfate, 1-5 g/L yeast powder, 5-10 g/L peptone and 1-5 g/L sucrose, and adding appropriate antibiotics, such as one or more of 30-200 μ g/mL ampicillin, 30-100 μ g/mL chloramphenicol and 30-200 μ g/mL kanamycin. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strain. Bacterial colonies on the plate form bacterial lawn, the whole plate thallus is inoculated into a seed shake flask, the seed shake flask culture medium is identical to the plate culture medium, shaking culture is carried out at 37 ℃ and 170rpm for 7h, and then a first-stage seeding tank is inoculated.
The inoculation amount of the first-stage seed tank is 0.1-1%, the ventilation amount is 0.4-0.6 vvm, the stirring speed is 300-600 rpm, the tank pressure is 0.05-0.08 MPa, and DO is controlled in the fermentation process>5%, the pH value during fermentation is controlled at 6.7-7.2, the culture is carried out for 16-18 h at 37 ℃, and the OD is 600 After the dilution reaches 0.8-1.0 (25 times), inoculating the mixture into a 10L seeding tank, wherein the culture medium of the seeding tank comprises 4-10 g/L monopotassium phosphate, 0.3-1 g/L magnesium sulfate heptahydrate, 2-6 g/L ammonium sulfate, 1-5 g/L yeast powder, 5-10 g/L peptone, 30-50 g/L glucose, 30-100 mu g/mL ampicillin and 30-100 mu g/mL chloramphenicol. Selecting suitable antibiotics, such as one or more of ampicillin 30-200. mu.g/mL, chloramphenicol 30-100. mu.g/mL, and kanamycin 30-200. mu.g/mL. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strain.
The fermentation process comprises the steps of inoculating 10-20%, ventilating 0.4-0.5 vvm, stirring at 800rpm and 0.05-0.1 MPa, controlling DO to be more than 20% in the fermentation process, controlling the pH value to be 6.7-7.2 in the fermentation process, feeding 50-70% of glucose solution in the fermentation process, controlling the residual sugar concentration of the fermentation liquor to be 0.5-1% and controlling the fermentation period to be 50-70 h. The fermentation medium comprises: 3-8 g/L potassium dihydrogen phosphate, 1-3 g/L magnesium sulfate, 0.01-0.03 g/L ferrous sulfate, 0.01-0.04 g/L manganese sulfate, 5-15 g/L ammonium sulfate, 20-40 g/L glucose, 5-30 g/L corn steep liquor, 0.5-3 g/L betaine and 0.002-0.006 g/L vitamin B1, and selecting appropriate antibiotics, such as one or more of 30-200 μ g/mL ampicillin, 30-100 μ g/mL chloramphenicol and 30-200 μ g/mL kanamycin. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strain.
Further, the glutaric acid content in the fermentation liquor is determined by adopting a gas chromatography, specifically, after the fermentation liquor is sampled, a centrifuge is used for centrifuging at 5000rpm for 5 minutes, and the supernatant is taken for determination. The gas chromatograph was GC2010pro (SHIMADZU, Japan), an autosampler, an amount of sample of 0.5 μ L, a split ratio of 20: 1. the inlet temperature is 290 ℃, the carrier gas flow rate is 1.2mL/min, and the detector FID set temperature is 320 ℃. The column was started at 180 ℃ for 2 minutes, warmed to 300 ℃ at a rate of 15 ℃ per minute and held for 7 minutes. The column model was Wondacap-5:30m 0.25mm 0.25. mu.m.
Further, the biomass of the cells was measured for the absorbance at 600nm by a spectrophotometer by OD 600 To indicate. The ammonia nitrogen concentration of the fermentation liquor is measured by adopting a conventional Kjeldahl method. The intermediate lysine in the fermentation broth was determined by conventional ninhydrin colorimetric method.
Furthermore, in the fermentation process, an online tail gas analyzer is used for detecting the content of carbon dioxide and oxygen in tail gas, and biostar software carried by FUS-30L advanced fermentation tank of national enhanced biochemical equipment company is used for acquiring process parameters such as DO, CER, OUR, RQ and the like on line.
Advantageous effects
The method is based on the application of the nitrogen source regulation strategy in the biological method glutaric acid guided by the on-line microbial physiological characteristic parameters OUR and CER in the fermentation process, and the method has the advantages of high glutaric acid yield, high saccharic acid conversion rate and easiness in large-scale amplification.
Drawings
FIG. 1 shows the deamination and decarboxylation reactions in the synthesis of glutaric acid based on the AMV pathway.
FIG. 2 shows that only 25% ammonia water is fed and the feeding rate of ammonia water is controlled so that OUR and CER in acid production period are 60-80 m mol L - 1 h -1 The glutaric acid real-time fermentation process parameter diagram.
FIG. 3 shows that 30% sodium hydroxide solution is used to regulate pH of fermentation broth and control 25% ammonia water flow rate, so that OUR and CER in acid production period are 120-140 m mol L -1 h -1 The glutaric acid real-time fermentation process parameter diagram.
FIG. 4 shows that 50% ammonium sulfate solution is fed in, and the feeding rate of ammonium sulfate is controlled so that OUR and CER in the early stage of acid production are kept at 180m mol L of 160- -1 h -1 The OUR and CER in the middle and later stages of acid production are maintained at 60-80 m mol L -1 h -1 The glutaric acid real-time fermentation process parameter diagram.
FIG. 5 shows the feeding of 50% ammonium sulfate solution, the feeding rate of ammonium sulfate is controlled to make the product yieldOUR and CER in the acid prophase are maintained at 140-160 m mol L -1 h -1 The OUR and CER in the middle and later stages of acid production are kept between 80 and 100m mol L -1 h -1 The glutaric acid real-time fermentation process parameter diagram.
FIG. 6 shows that 50% ammonium sulfate solution is fed-batch, and the feeding-batch speed of ammonium sulfate is controlled so that OUR and CER in acid production period are respectively 60-80 mmol L -1 h -1 And 80-10 m mol L -1 h -1 The glutaric acid production rate curve and the thallus growth curve chart in the fermentation process of the two schemes.
FIG. 7 shows that 50% ammonium sulfate solution is fed-batch, and the feeding-batch rate of ammonium sulfate is controlled so that OUR and CER from the acid production period to the end of fermentation are maintained at 160-180 m mol L -1 h -1 The glutaric acid real-time fermentation process parameter diagram.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention can be made by those skilled in the art after reading the teaching of the present invention, and these equivalents also fall within the scope of the claims appended to the present application.
Example 1
Feedback regulation and control of ammonia water flow acceleration based on microbial physiological parameters OUR and CER
The metabolic engineering strain Escherichia coli LQ-1 constructed in the laboratory is adopted to carry out glutaric acid fermentation, the construction method reference is carried out by a conventional method, plasmids of a lysine 2-monooxygenase (DavB) encoding gene and a delta-aminopentanamide enzyme (DavA) encoding gene from pseudomonas putida are transferred into host bacteria, and 5-aminopentanoate aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD) are further overexpressed by the plasmids:
(1) after the glycerol tube is naturally thawed, 300uL of glycerol tube is taken out by a sterilized gun head and laid on a plate culture medium as much as possible, and the glycerol tube is inversely cultured for 24 hours in a constant-temperature incubator at 37 ℃. The activation medium is: 4g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 4.5g/L ammonium sulfate, 5g/L yeast powder, 8g/L peptone, 3g/L sucrose, 50 mu g/mL ampicillin and 50 mu g/mL chloramphenicol.
(2) The bacterial colony on the plate forms bacterial lawn, the whole plate thallus is taken by an inoculating loop to a seed shaking flask, after shaking culture is carried out for 7 hours at 37 ℃ and 170rpm, the plate thallus is inoculated to a first-stage seeding tank. The formula of the shake flask culture medium is the same as that of a plate culture medium.
(3) The seeding tank process comprises the following steps: the inoculation amount is 0.1 percent, the ventilation amount is 0.4vvm, the stirring rotation speed is 300-600 rpm, the tank pressure is 0.05-0.08 MPa, and DO is controlled in the culture process>5% and 25% ammonia water, controlling pH value of fermentation process to 6.7, culturing at 37 deg.C for 16-18 h, OD 600 After reaching 0.8-1.0 (diluted 25 times), the mixture was inoculated into a 30L fermentor. The first-level seeding tank culture medium is as follows: 6g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 3g/L ammonium sulfate, 4g/L yeast powder, 6g/L peptone, 40g/L glucose, 50 mu g/mL ampicillin and 50 mu g/mL chloramphenicol.
(4) The fermentation process comprises the following steps: the inoculation amount is 14 percent, the stirring speed is related to DO, so that DO 20-40 percent, the tank pressure is 0.05-0.1 MPa, the ventilation volume is 0.4-0.6 vvm, the fermentation temperature is controlled to be 37 ℃, the pH value in the fermentation process is maintained to be 6.7, in the fermentation process, 70 percent of glucose solution is fed, the residual sugar concentration in the fermentation liquid is controlled to be 0.5-1 percent, and the fermentation period is 60 hours. Fermentation medium: magnesium sulfate heptahydrate 1.6g/L, ferrous sulfate heptahydrate 0.03g/L, manganese sulfate monohydrate 0.032g/L, ammonium sulfate 10g/L, potassium dihydrogen phosphate 5g/L, glucose 30g/L, corn steep liquor 25g/L, betaine 2.2g/L, vitamin B1 0.006g/L, ampicillin 50 mug/mL, chloramphenicol 50 mug/mL.
Two ammonia flowadd strategies were employed:
inoculating the seeds into a 30L fermentation tank for fermentation, and fermenting when OUR and CER reach 140m mol L -1 h -1 When in use, ammonia water feeding is started, and the feeding speed of the ammonia water is kept between 60 and 80m mol L according to the real-time OUR and CER -1 h -1 ；
Inoculating the seeds into a 30L fermentation tank for fermentation, and fermenting when OUR and CER reach 140m mol L -1 h -1 When in use, ammonia water flow addition is started, the pH value of the fermentation liquor is regulated and controlled by 30% sodium hydroxide solution, and the flow addition speed of the ammonia water is according to real-time OUR and CER, so thatThe OUR and CER in the acid production period are kept at 120- -1 h -1 The real-time parameters of the fermentation process for the two schemes are shown in figure 1 and figure 2. The fermentation was carried out for 60h and the results are shown in Table 1.
TABLE 1 comparison of glutaric acid fermentation results corresponding to different control strategies
Example 2
Ammonium sulfate solution flow acceleration is feedback-regulated and controlled based on microbial physiological parameters OUR and CER
The procedure and method of example 1 were followed except that 50% ammonium sulfate was fed-batch used to provide a nitrogen source for glutaric acid synthesis and 30% sodium hydroxide solution was used to control the pH of the fermentation broth. The nitrogen source flow acceleration is subjected to feedback regulation according to real-time OUR and CER, and three schemes are adopted:
scheme1 inoculating the seeds into a 30L fermentation tank for fermentation, and when the OUR and CER reach 140m mol L -1 h -1 When in use, ammonia water feeding is started, and the feeding speed of the ammonium sulfate solution is kept at 160-180 m mol L according to the real-time OUR and CER -1 h -1 The OUR and CER in the middle and later stages of acid production are maintained at 60-80 m mol L -1 h -1 。
Scheme2 inoculating the seeds into a 30L fermentation tank for fermentation, and when the OUR and CER reach 140m mol L -1 h -1 When in use, ammonia water feeding is started, and the feeding speed of the ammonium sulfate solution is kept at 140-160 m mol L according to the real-time OUR and CER -1 h -1 The OUR and CER in the middle and later stages of acid production are kept between 80 and 100m mol L -1 h -1 。
Scheme3 inoculating the seeds into a 30L fermentation tank for fermentation, and when the OUR and the CER reach 140m mol L -1 h -1 When in use, ammonia water feeding is started, and the feeding speed of the ammonium sulfate solution is kept at 160-180 m mol L according to the real-time OUR and CER -1 h -1 。
The real-time parameters of the fermentation process of the three schemes are shown in figures 4, 5 and 7. As shown in FIGS. 4, 5 and 6, when the high OUR and CER are used to feedback control the nitrogen source feeding rate, the glutaric acid production rate is faster. And (3) regulating and controlling the nitrogen source flow acceleration according to Scheme3, and fermenting for 60 hours, wherein the yield of glutaric acid and the saccharic acid conversion rate in the fermentation liquid are the highest, and are 53.65g/L and 46.76% respectively.
Claims (10)
1. A method for producing glutaric acid by performing feedback regulation and control on nitrogen source feeding based on microbial physiological parameters comprises the following steps:
inoculating the strain into a fermentation tank containing a fermentation medium, and performing feedback regulation and control on nitrogen source feeding according to real-time microbial physiological parameters to obtain glutaric acid through fermentation;
wherein the microorganism physiological parameters comprise oxygen consumption rate OUR and carbon dioxide generation rate CER.
2. The method as claimed in claim 1, wherein the OUR and CER rise to 140-180m mol L as the fermentation progresses -1 h -1 At this time, the nitrogen source feed was started.
3. The method of claim 1, wherein the regulated nitrogen source fedbatch is a regulated nitrogen source fedbatch rate that is automatically feedback adjusted based on real-time OUR, CER, or manually feedback adjusted based on real-time OUR, CER.
4. The process as claimed in claim 3, wherein OUR and CER are less than 140-180m mol L during fermentation -1 h -1 Increasing the nitrogen source flow acceleration rate to maintain OUR and CER at 140- -1 h -1 。
5. The method of claim 1, wherein the nitrogen source is one or more of ammonia water, liquid ammonia, ammonium sulfate solution, ammonium chloride solution, urea solution, ammonium carbonate and ammonium bicarbonate solution.
6. The method of claim 1, wherein the fermentation further comprises feeding a glucose solution such that the concentration of glucose in the fermentation broth is 0.5-1% (w/v).
7. The method of claim 6, wherein the fed-in glucose solution has a concentration of 50-70% (w/v).
8. The method of claim 1, wherein the fermentation process parameters comprise: the inoculation amount is 10-20%, the fermentation temperature is 35-37 ℃, the pH value of the fermentation liquor is controlled to be 6.7-7.2, the stirring speed is related to dissolved oxygen DO, the DO is maintained at 20-40%, the tank pressure is kept at 0.05-0.1 MPa, and the fermentation period is 50-70 h.
9. The method of claim 1, wherein the fermentation medium comprises: 3-8 g/L monopotassium phosphate, 1-3 g/L magnesium sulfate, 0.01-0.03 g/L ferrous sulfate, 0.01-0.04 g/L manganese sulfate, 5-15 g/L ammonium sulfate, 20-40 g/L glucose, 5-30 g/L corn steep liquor, 0.5-3 g/L betaine, 0.002-0.006 g/L vitamin B1 and antibiotics added into the culture medium, wherein the antibiotics are one or more of 30-200 mu g/mL ampicillin, 30-100 mu g/mL chloramphenicol and 30-200 mu g/mL kanamycin.
10. The method of claim 1, wherein the bacterial species is metabolically engineered escherichia coli or corynebacterium glutamicum.
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| CN115948622A (en) * | 2022-09-22 | 2023-04-11 | 北京蓝晶微生物科技有限公司 | Microbial fermentation control method, device, system, equipment and medium |
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