CN114921502B - Glutaric acid production method for feedback regulation and control of nitrogen source flow based on microorganism physiological parameters - Google Patents

Abstract

The application relates to a glutaric acid production method based on feedback regulation and control of nitrogen source flow based on microorganism physiological parameters, which comprises the steps of inoculating strain culture solution into a fermentation tank containing a fermentation culture medium, and fermenting to obtain glutaric acid by using real-time microorganism physiological parameters oxygen consumption rate (OUR) and carbon dioxide generation rate (CER) in the fermentation process as the basis of feedback regulation and control of nitrogen source flow acceleration. The application has high acid production and conversion rate, is easy to scale up and lays a foundation for industrialization of biological glutaric acid.

Description

A glutaric acid production method based on feedback control of nitrogen source feeding based on microbial physiological parameters production method

Technical field

The invention belongs to the field of biology, and in particular relates to a glutaric acid production method based on feedback control of nitrogen source feeding based on microbial physiological parameters.

Background technique

Glutaric acid can be synthesized into glutaric anhydride, which is widely used in rubber, medicine and other fields. In addition, new polyamides can be obtained by polymerizing glutaric acid with different diamines. Currently, most of the glutaric acid on the market is obtained by separating the by-products of adipic acid or through chemical reactions. Chemical synthesis of glutaric acid requires high temperature, high pressure, and expensive catalysts. For example, glutaric acid can be synthesized by ring-opening butyrolactone with the highly toxic compound potassium cyanide, and then performing a hydrolysis reaction. This process is highly toxic, costly, and process-safe. Low and environmentally unfriendly. Obtaining high-purity glutaric acid by separating the by-products of adipic acid requires multi-step crystallization, low yield, and high cost, which limits the further application of glutaric acid. In recent years, metabolic engineering of microorganisms to produce glutaric acid has become a hot research topic, and microorganisms including Escherichia coli and Corynebacterium glutamicum have been widely developed to synthesize glutaric acid. The 5-aminovaleric acid pathway (AMV) pathway is considered the most promising glutaric acid biosynthetic pathway. As shown in Figure 1, the AMV pathway is achieved by selecting a suitable host, such as Escherichia coli or Corynebacterium glutamicum, in which the lysine 2-monooxygenase derived from Pseudomonas putida is overexpressed. (DavB) and δ-aminovaleramidase (DavA), metabolize lysine into 5-aminovaleric acid, which is further metabolized by 5-aminovaleric acid aminotransferase (GabT) and succinate semialdehyde dehydrogenase (GabD) ) to obtain glutaric acid. So far, research on the biological synthesis of glutaric acid has mainly focused on the metabolic engineering of microorganisms, while little research has been done on the fermentation process of glutaric acid. Microbial production of industrial L-lysine requires large amounts of nitrogen sources, such as ammonium sulfate and liquid ammonia, while glutaric acid production based on L-lysine catabolism requires stepwise deamination and transamination reactions. Therefore, the nitrogen source fed-batch strategy will interfere with the synthesis of L-lysine and glutaric acid. However, there is no relevant literature reporting how the nitrogen source affects the synthesis of glutaric acid. Also shown in Figure 1, L-lysine to 5-aminovaleramide requires oxygen and produces carbon dioxide, which is related to the real-time physiological parameters of microbial oxygen uptake rate (OUR) and carbon dioxide release rate (CER). In the present invention, we developed a nitrogen source feeding strategy based on feedback regulation of real-time microbial physiological parameters, and applied it to the biological production of glutaric acid. This process has high acid production and high sugar-acid conversion rate, and can be easily scaled up to industrial scale.

Contents of the invention

In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is to provide a glutaric acid production method that uses feedback control of nitrogen source feeding based on microbial physiological parameters.

The present invention provides a glutaric acid production method based on feedback control of nitrogen source feeding based on microbial physiological parameters, including:

The bacteria are inoculated into the fermentation tank containing the fermentation medium, and the nitrogen source is fed back and regulated based on real-time microbial physiological parameters to ferment to obtain glutaric acid. Among them, the microbial physiological parameters include oxygen consumption rate (OUR) and carbon dioxide production. rate (CER).

Further, the culture solution of glutaric acid-producing bacteria is inoculated into the fermentation tank containing the fermentation medium. Under the appropriate medium and culture conditions, the microorganisms use the medium to grow. When the real-time OUR and CER gradually rise to 140-180m molL ^-1 h ^-1 , the microorganism enters the rapid synthesis stage of glutaric acid. At this time, the nitrogen source feeding is started.

The control of nitrogen source flow addition is to regulate the speed of nitrogen source flow addition. The speed of nitrogen source flow addition can be automatically adjusted based on real-time OUR and CER feedback, or can be manually adjusted based on real-time OUR and CER.

Further, when the OUR and CER during the acid production period are lower than 140-180m mol L ^-1 h ^-1 , the nitrogen source flow acceleration is increased to keep the OUR and CER at 140-180m mol L ^-1 h ^-1 , more preferably 160 –180m mol L ^-1 h ^-1 .

The nitrogen source is one or more of ammonia, liquid ammonia, ammonium sulfate solution, ammonium chloride solution, urea solution, ammonium carbonate, ammonium bicarbonate solution, preferably ammonium sulfate solution, wherein the solution is aqueous solution.

When using ammonia water for nitrogen source flow, you can use ammonia water to adjust the pH value of the fermentation liquid at the same time to keep the pH value at 6.7-7.2. You can also use sodium hydroxide solution to control the pH value of the fermentation liquid. The concentration of ammonia water is generally between 25–28% (w/v), the concentration of sodium hydroxide solution is generally 5–40% (w/v).

Further preferably, 10-50% (w/v) ammonium sulfate solution is fed to provide a nitrogen source, and 5-40% (w/v) sodium hydroxide solution is added to regulate the pH value of the fermentation broth to 6.7-7.2.

The fermentation process also includes feeding glucose solution so that the concentration of glucose in the fermentation broth is 0.5-1% (w/v). The glucose solution is fed, wherein the concentration of the glucose solution is 50-70% (w/v).

The fermentation process parameters include: inoculation amount 10-20%, fermentation temperature 35-37°C, controlling the pH value of the fermentation liquid to 6.7-7.2, and linking the stirring speed to dissolved oxygen (DO) to maintain DO at 20-40%. The tank pressure is maintained at 0.05–0.1MPa, and the fermentation cycle is 50–70h.

The culture medium used for the fermentation is: potassium dihydrogen phosphate 3-8g/L, 1-3g/L magnesium sulfate, 0.01-0.03g/L ferrous sulfate, 0.01-0.04g/L manganese sulfate, 5-15g/L L ammonium sulfate, 20-40g/L glucose, 5-30g/L corn steep liquor, 0.5-3g/L betaine, 0.002-0.006g/L vitamin B1, antibiotics are added to the culture medium, wherein the antibiotic is 30-200μg /mL ampicillin, 30-100μg/mL chloramphenicol, 30-200μg/mL kanamycin or one or more.

The bacterial strain is metabolically engineered Escherichia coli or Corynebacterium glutamicum.

Note: The mass volume percentage (w/v) in the present invention is all g/100ml.

Furthermore, the glutaric acid-producing bacteria used in this experiment are metabolically engineered strains. There are many reports on glutaric acid metabolism engineering bacteria. The modification of glutaric acid-producing bacteria based on the AMV pathway mainly involves the encoding of lysine 2-monooxygenase (DavB) and δ-aminovaleramidase (DavA) from Pseudomonas putida. The gene plasmid is transferred into the host bacteria, and 5-aminovalerate aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD) are further overexpressed through the plasmid to obtain glutaric acid. Lysine is used to synthesize glutaric acid. Precursor, therefore enhancing the metabolic flux of lysine is also a common method. At present, glutaric acid-producing bacteria are mostly modified Escherichia coli or Corynebacterium glutamicum. For example, the research report of Han et al. Glutaric acid production by systems metabolic engineering of an L-lysine–overproducing Corynebacterium glutamicum(2020,Pnas.117,30328-30334), Kim et al.’s research report Metabolic engineering of Corynebacterium glutamicum for theproduction of glutaric acid,a C5 dicarboxylic acid platformchemical(2019,Metab.Eng. 51,99–109), Li et al.’s research report Targeting metabolic driving and intermediate influx in L-lysine catabolism for high-level glutarateproduction. (2019, Nat. Commun. 10, 3337), Rohles et al. Systems metabolic engineering of Corynebacterium glutamicum for the production of the carbon-5platform chemicals 5-aminovalerate and glutarate. (2016, Microb.Cell.Fact.15,1-13), Adkins et al. Research report Engineering Escherichia coli for RenewableProduction of the 5-Carbon Polyamide Building-Blocks 5-Aminovalerate and Glutarate (2013, Biotechnol. Bioeng. 110, 1726-1734).

Further, this process will be explained by taking the metabolic engineering strain Escherichia coli LQ-1 constructed by the inventor's laboratory based on the AMV pathway and referring to the above-mentioned literature to synthesize glutaric acid as an example to illustrate this process. The claims are not limited to the laboratory strains and related culture media. and cultivation methods.

Biological glutaric acid fermentation includes seed activation in glycerol tubes, seed culture in shake flasks, seed culture in seed tanks and fermentation processes.

After the glycerin tube is naturally thawed, use a sterilized pipette tip to take 300uL on the plate culture medium, spread it as much as possible, and incubate it upside down in a 37°C constant temperature incubator for 24 hours. The plate culture medium is: 4–10g/L potassium dihydrogen phosphate, 0.3– 1g/L magnesium sulfate heptahydrate, 2–6g/L ammonium sulfate, 1–5g/L yeast powder, 5–10g/L peptone, 1–5g/L sucrose, add appropriate antibiotics, such as 30–200μg/mL ampicillin One or more of penicillin, 30–100 μg/mL chloramphenicol, and 30–200 μg/mL kanamycin. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strain. The bacterial colonies on the plate form a bacterial lawn. Use an inoculating loop to take the entire bacterial plate and put it into a seed shake flask. The seed shake flask culture medium is the same as the plate culture medium. After culturing for 7 hours on a shaking table at 37°C and 170 rpm, inoculate the first-level seed tank.

The inoculum volume of the first-level seed tank is 0.1–1%, the ventilation volume is 0.4–0.6vvm, the stirring speed is 300–600rpm, the tank pressure is 0.05–0.08MPa, the DO is controlled to be >5% during the fermentation process, and the pH value during the fermentation process is controlled at 6.7–7.2. , 37℃, culture for 16-18h, after the OD ₆₀₀ reaches 0.8-1.0 (diluted 25 times), inoculate into a 10L seed tank. The seed tank culture medium is 4-10g/L potassium dihydrogen phosphate, 0.3-1g/L Magnesium sulfate heptahydrate, 2–6g/L ammonium sulfate, 1–5g/L yeast powder, 5–10g/L peptone, 30–50g/L glucose, 30–100μg/mL ampicillin, 30–100μg/mL chloramphenicol white. Choose an appropriate antibiotic, such as one or more of 30–200 μg/mL ampicillin, 30–100 μg/mL chloramphenicol, and 30–200 μg/mL kanamycin. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strain.

The fermentation process is as follows: inoculation amount 10–20%, ventilation volume 0.4–0.5vvm, stirring speed 300–800rpm, tank pressure 0.05–0.1MPa, controlling DO>20% during the fermentation process, controlling the pH value during the fermentation process 6.7–7.2, and controlling the fermentation process. , add 50-70% glucose solution, and control the residual sugar concentration of the fermentation broth to 0.5-1%, and the fermentation cycle is 50-70h. Fermentation medium includes: potassium dihydrogen phosphate 3–8g/L, 1–3g/L magnesium sulfate, 0.01–0.03g/L ferrous sulfate, 0.01–0.04g/L manganese sulfate, 5–15g/L ammonium sulfate, 20–40g/L glucose, 5–30g/L corn steep liquor, 0.5–3g/L betaine, 0.002–0.006g/L vitamin B1, choose appropriate antibiotics, such as 30–200μg/mL ampicillin, 30–100μg/ mL chloramphenicol, 30-200 μg/mL kanamycin, or one or more. The addition of antibiotic species depends on the antibiotic resistance characteristics of the metabolically engineered strain.

Further, gas chromatography was used to determine the glutaric acid content in the fermentation broth. Specifically, after sampling the fermentation broth, centrifuge at 5000 rpm for 5 minutes, and take the supernatant for measurement. The gas chromatograph is GC2010pro (SHIMADZU, Japan), automatic sampler, injection volume 0.5 μL, split ratio 20:1. The inlet temperature is 290°C, the carrier gas flow rate is 1.2mL/min, and the detector FID setting temperature is 320°C. The initial temperature of the chromatographic column was 180°C, maintained for 2 minutes, raised to 300°C at a rate of 15°C per minute, and maintained for 7 minutes. The column model is Wondacap-5:30m◇0.25mm◇0.25μm.

Furthermore, the bacterial biomass was measured by the absorbance value at 600 nm using a spectrophotometer, and expressed as OD ₆₀₀ . The ammonia nitrogen concentration of the fermentation broth was determined by the conventional Kjeldahl nitrogen determination method. The intermediate lysine in the fermentation broth was determined by the conventional ninhydrin colorimetric method.

Furthermore, during the fermentation process, an online exhaust gas analyzer was used to detect the carbon dioxide and oxygen content in the exhaust gas, and the biostar software included in the FUS-30L advanced fermentation tank of Guoqiang Biochemical Equipment Company was used to collect process parameters such as DO, CER, OUR, and RQ online. .

beneficial effects

The present invention is based on the application of nitrogen source control strategy guided by online microbial physiological characteristic parameters OUR and CER in the fermentation process in biological glutaric acid. This method has high glutaric acid yield, high sugar acid conversion rate, and is easy to scale up. enlarge.

Description of the drawings

Figure 1 shows the deamination and decarboxylation reactions in the glutaric acid synthesis process based on the AMV pathway.

Figure 2 is a parameter diagram of glutaric acid real-time fermentation process in which only 25% ammonia water is added and the ammonia water flow acceleration is controlled so that the OUR and CER in the acid production period are 60–80m mol L ^{- 1} h ^-1 .

Figure 3 is a parameter diagram of glutaric acid real-time fermentation process using 30% sodium hydroxide solution to adjust the pH of the fermentation broth and control the acceleration of 25% ammonia water flow so that the OUR and CER during the acid production period are 120–140m mol L ^-1 h ^-1 .

Figure 4 shows the feeding of 50% ammonium sulfate solution, and the ammonium sulfate flow acceleration is controlled so that OUR and CER are maintained at 160–180m mol L ^-1 h ^-1 in the early stage of acid production, and OUR and CER are maintained at 60–80m mol in the middle and late stages of acid production. L ^-1 h ^-1 glutaric acid real-time fermentation process parameter diagram.

Figure 5 shows the feeding of 50% ammonium sulfate solution, and the ammonium sulfate flow acceleration is controlled so that OUR and CER are maintained at 140–160m mol L ^-1 h ^-1 in the early stage of acid production, and OUR and CER are maintained at 80–100m mol in the middle and late stages of acid production. L ^-1 h ^-1 glutaric acid real-time fermentation process parameter diagram.

Figure 6 shows the two scenarios of feeding 50% ammonium sulfate solution and controlling the ammonium sulfate flow acceleration so that the acid production period OUR and CER are 60–80mmol L ^-1 h ^-1 and 80–10m mol L ^-1 h ^-1 respectively. Glutaric acid acid production rate curve and bacterial growth curve during the fermentation process.

Figure 7 is a parameter diagram of glutaric acid real-time fermentation process in which 50% ammonium sulfate solution is added and the ammonium sulfate flow acceleration is controlled to maintain the OUR and CER at 160–180m mol L ^-1 h ^-1 from the acid production period to the end of fermentation.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Example 1

Feedback control of ammonia water flow acceleration based on microbial physiological parameters OUR and CER

The metabolic engineering strain Escherichia coli LQ-1 constructed in our laboratory was used for glutaric acid fermentation. The construction method was carried out by conventional methods according to the literature, mainly by adding lysine 2-monooxygenase from Pseudomonas putida. The plasmids encoding genes for (DavB) and δ-aminovaleramidase (DavA) were transferred into the host bacteria, and 5-aminovalerate aminotransferase (GabT) and succinate semialdehyde dehydrogenase (GabT) were further overexpressed through the plasmids. GabD. The specific fermentation process is as follows:

(1) After the glycerol tube is naturally thawed, use a sterilized pipette tip to take 300uL on the plate culture medium, spread it out as much as possible, and incubate it upside down in a 37°C constant-temperature incubator for 24 hours. 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, 50μg/mL ampicillin , 50μg/mL chloramphenicol.

(2) The bacterial colonies on the plate form a bacterial lawn. Use an inoculating loop to take the entire bacterial plate and place it in a seed shaker flask. After culturing on a shaker at 37°C and 170rpm for 7 hours, inoculate the first-level seed tank. The formula of shake flask culture medium is the same as that of plate culture medium.

(3) Seed tank technology: inoculation amount 0.1%, ventilation volume 0.4vvm, stirring speed 300–600rpm, tank pressure 0.05–0.08MPa, control DO>5% during the culture process, 25% ammonia water controls the pH value of the fermentation process to 6.7, Incubate at 37°C for 16-18 hours. After the OD ₆₀₀ reaches 0.8-1.0 (diluted 25 times), inoculate it into a 30L fermenter. The first-level seed tank culture medium is: 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, 50μg/mL Ampicillin, 50μg/mL chloramphenicol.

(4) Fermentation process: the inoculation amount is 14%, the stirring speed is related to DO, so that during the fermentation process, DO is 20%–40%, the tank pressure is 0.05–0.1MPa, the ventilation volume is 0.4–0.6vvm, and the fermentation temperature is controlled to 37°C. The pH value is maintained at 6.7. During the fermentation process, 70% glucose solution is added, and the residual sugar concentration of the fermentation liquid is controlled at 0.5-1%. The fermentation cycle is 60 hours. Fermentation medium: 1.6g/L magnesium sulfate heptahydrate, 0.03g/L ferrous sulfate heptahydrate, 0.032g/L manganese sulfate monohydrate, 10g/L ammonium sulfate, 5g/L potassium dihydrogen phosphate, 30g/L glucose , 25g/L corn steep liquor, 2.2g/L betaine, 0.006g/L vitamin B1, 50μg/mL ampicillin, 50μg/mL chloramphenicol.

Two ammonia water addition strategies are used:

The seeds are connected to the 30L fermentation tank for fermentation. When OUR and CER reach 140m mol L ^-1 h ^-1 , the ammonia water flow is started. The ammonia flow acceleration is based on the real-time OUR and CER, so that the OUR and CER during the acid production period are maintained at 60 –80m mol L ^-1 h ^-1 ;

The seeds are connected to the 30L fermentation tank for fermentation. When OUR and CER reach 140m mol L ^-1 h ^-1 , the ammonia flow is started to be added, and 30% sodium hydroxide solution is used to adjust the pH value of the fermentation liquid. The ammonia flow acceleration is based on the real-time OUR and CER, so that OUR and CER during the acid production period are maintained at 120–140m mol L ^-1 h ^-1 . The real-time parameters of the fermentation process of the two schemes are shown in Figures 1 and 2. The fermentation was carried out for 60 hours, and the fermentation results are shown in Table 1.

Table 1 Comparison of glutaric acid fermentation results corresponding to different control strategies

Example 2

Feedback control of ammonium sulfate solution flow acceleration based on microbial physiological parameters OUR and CER

According to the process and method of Example 1, the difference is that 50% ammonium sulfate is used to provide a nitrogen source for glutaric acid synthesis, and 30% sodium hydroxide solution is used to control the pH value of the fermentation broth. The nitrogen source flow acceleration is feedback adjusted based on the real-time OUR and CER. There are three options:

Scheme1: The seeds are connected to the 30L fermentation tank for fermentation. When OUR and CER reach 140m mol L ^-1 h ^-1 , the ammonia water flow is started. The flow acceleration of the ammonium sulfate solution is based on the real-time OUR and CER, so that the OUR and CER in the early stage of acid production are CER was maintained at 160–180m mol L ^-1 h ^-1 , and OUR and CER were maintained at 60–80m mol L ^-1 h ^-1 in the middle and late stages of acid production.

Scheme 2: The seeds are connected to the 30L fermenter for fermentation. When OUR and CER reach 140m mol L ^-1 h ^-1 , the ammonia water flow is started. The flow acceleration of the ammonium sulfate solution is based on the real-time OUR and CER, so that the OUR and CER in the early stage of acid production are CER was maintained at 140–160m mol L ^-1 h ^-1 , and OUR and CER were maintained at 80–100m mol L ^-1 h ^-1 in the middle and late stages of acid production.

Scheme 3: The seeds are connected to the 30L fermentation tank for fermentation. When OUR and CER reach 140m mol L ^-1 h ^-1 , the ammonia water flow is started. The flow acceleration of the ammonium sulfate solution is based on the real-time OUR and CER, so that the acid production period OUR and The CER remains at 160–180m mol L ^-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 Figures 4, 5, and 6, when high OUR and CER are used to feedback control the nitrogen source flow acceleration, glutaric acid produces acid at a faster rate. The nitrogen source flow acceleration was controlled according to Scheme 3, and the fermentation was carried out for 60 hours. The glutaric acid yield and sugar acid conversion rate in the fermentation broth were the highest, which were 53.65g/L and 46.76% respectively.

Claims (6)

1. A glutaric acid production method for feedback regulation and control of nitrogen source flow based on microbial physiological parameters, comprising:

inoculating the strain into a fermentation tank containing a fermentation medium, and carrying out fermentation by using a real-time microbial physiological parameter as a basis to feed back and regulate nitrogen source flow so as to obtain glutaric acid; the strain is metabolic engineering escherichia coli;

wherein the microbial physiological parameter comprises an oxygen consumption rate OUR and a carbon dioxide generation rate CER; wherein OUR and CER rise to 140-180 mmmol L as fermentation proceeds ^-1 h ^-1 In this case, nitrogen source feeding is started, and OUR and CER in the fermentation process are lower than 140m mol L ^-1 h ^-1 Increasing acceleration of nitrogen source flow to keep OUR and CER at 140-180 mmmol L ^-1 h ^-1 The method comprises the steps of carrying out a first treatment on the surface of the The fermentation process also comprises feeding glucose solution so that the concentration of glucose in the fermentation liquid is 0.5-1% (w/v).

2. The method of claim 1, wherein the regulating nitrogen flow is regulating a speed of nitrogen flow, and nitrogen flow acceleration is based on real-time OUR, CER automatic feedback adjustment, or manual feedback adjustment based on real-time OUR, CER.

3. The method according to 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 solution, ammonium bicarbonate solution.

4. The method according to claim 1, wherein the glucose solution is fed in a concentration of 50-70% (w/v).

5. The method of claim 1, the fermentation process parameters comprising: the inoculation amount is 10-20%, the fermentation temperature is 35-37 ℃, the pH value of the fermentation liquid is controlled to be 6.7-7.2, the stirring speed is related to dissolved oxygen DO, so that DO is maintained at 20-40%, the tank pressure is maintained at 0.05-0.1MPa, and the fermentation period is 50-70h.

6. The method of claim 1, wherein the fermentation medium comprises: 3-8g/L potassium dihydrogen phosphate, 1-3g/L magnesium sulfate, 0.01-0.03g/L ferrous sulfate, 0.01-0.04g/L manganese sulfate, 5-15g/L ammonium sulfate, 20-40g/L glucose, 5-30g/L corn steep liquor, 0.5-3g/L betaine, 0.002-0.006g/L vitamin B1 and one or more antibiotics are added into the culture medium, wherein the antibiotics are 30-200 mug/mL ampicillin, 30-100 mug/mL chloramphenicol and 30-200 mug/mL kanamycin.