A fully synthetic medium for the propagation of 2-keto-gluconic acid producing bacteria and use thereof

Abstract

The application provides a full-synthetic culture medium for 2-keto-gluconic acid producing bacteria proliferation and application thereof, and belongs to the technical field of microorganisms. The full-synthetic culture medium for 2-keto-gluconic acid producing bacteria proliferation culture comprises 10.0-40.0 g / L of glucose, 0.5-4.0 g / L of urea, 5.0-30.0 mg / L of thiamine, 0.5-3.0 mg / L of biotin, 1.0-4.0 g / L of NaH2PO4, 0.7-2.1 g / L of KH2PO4, 0.1-0.3 g / L of MgSO4·7H2O and 0.0-5.0 g of CaCO3. The full-synthetic culture medium or semi-synthetic culture medium is subjected to proliferation culture, and it is shown that the full-synthetic culture medium can completely replace the semi-synthetic culture medium, guarantees the repeatability of culture results, and further fermentation production of the bacteria strains subjected to the proliferation culture will not affect the stability of the fermentation production of the bacteria strains.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a full-synthetic culture medium for proliferation of 2-keto-gluconic acid producing bacteria and application thereof. BACKGROUND

[0002] 2-keto-gluconic acid is an optically active organic acid formed by oxidation of glucose (biological oxidation or chemical oxidation), is one of the skeleton materials for constructing chiral compounds and heterocyclic compounds, and is currently most important for industrial use as a precursor for synthesis of antioxidant D-erythorbic acid and salts thereof. In addition, 2-keto-gluconic acid can also be directly used as a cement plasticizer, a cement retarder, a detergent builder, and an important component of calcium-rich feed additives.

[0003] Currently, common 2-keto-gluconic acid production methods can be roughly divided into four categories, including chemical oxidation method, enzyme catalytic oxidation method, microbial fermentation method, and microbial resting cell conversion method. Among them, the microbial fermentation method is currently the most economical, efficient and environmentally friendly 2-keto-gluconic acid industrial production method. In nature, there are many strains capable of fermenting glucose / gluconic acid to 2-keto-gluconic acid, which are usually derived from Pseudomonas, Erwinia, Acetobacter, Serratia, Gluconobacter, etc. Among them, Pseudomonas has become a commonly used strain for industrial production of 2-keto-gluconic acid at home and abroad due to its high sugar acid conversion efficiency and wide environmental adaptability.

[0004] The expansion culture (proliferation) of the strain is one of the key steps for fermentation production of the target product. However, the culture medium currently used for culture of 2-keto-gluconic acid producing bacteria is a semi-synthetic culture medium, and the nitrogen source thereof is mainly animal and plant extracts such as corn syrup, yeast extract or protein peptone, etc., which have unclear or inconsistent components, thereby affecting the repeatability of the strain culture results, and further affecting the stability of the subsequent fermentation of the producing bacteria. SUMMARY

[0005] Therefore, the purpose of the present application is to provide a full-synthetic culture medium for proliferation of 2-keto-gluconic acid producing bacteria, wherein the components are determined, and the preparation is facilitated, and the use of the culture medium for culture not only ensures the repeatability of the proliferation of 2-keto-gluconic acid producing bacteria, but also does not affect the stability of the subsequent fermentation of the producing bacteria.

[0006] The present application provides a full-synthetic culture medium for proliferation culture of 2-keto-gluconic acid producing bacteria, which comprises the following components per liter of culture medium:

[0007] The ingredients are: glucose 10.0–40.0 g, nitrogen source 0.5–4.0 g, thiamine 5.0–30.0 mg, biotin 0.5–3.0 mg, NaH2PO4 1.0–4.0 g, KH2PO4 0.7–2.1 g, MgSO4·7H2O 0.1–0.3 g, CaCO3 0.0–5.0 g, and pH 6.0–7.0.

[0008] Preferably, each liter of culture medium comprises the following components:

[0009] The concentrations were: glucose 20.0 g / L, nitrogen source 2.0 g / L, thiamine 17.5 mg / L, biotin 1.5 mg / L, NaH2PO4 2.1 g / L, KH2PO4 1.4 g / L, MgSO4·7H2O 0.2 g / L, CaCO3 5.0 g / L, and pH 6.7.

[0010] Preferably, the nitrogen source includes at least one of the following: urea, ammonium chloride, ammonium nitrate, ammonium sulfate, or sodium nitrate.

[0011] This invention provides the application of the fully synthetic culture medium in the proliferation culture of 2-ketogluconic acid-producing bacteria.

[0012] Preferably, the 2-ketogluconic acid producing bacteria includes at least one of the following: microbial strains of Pseudomonas, Erwinia, Acetobacter, Serratia, and Gluconobacter.

[0013] Preferably, the Pseudomonas genus includes at least one of the following: Pseudomonas plecoglossicida, Pseudomonas putida, and Pseudomonas fluorescens.

[0014] This invention provides a combined culture medium for the stable production of 2-ketogluconic acid, comprising the total synthetic culture medium and the fermentation culture medium;

[0015] The fermentation medium comprises 160–180 g / L glucose, 18–22 g / L corn steep liquor, 43–47 g / L CaCO3, and a pH of 6.6–6.8.

[0016] Preferably, the fermentation medium comprises 180 g / L glucose containing one molecule of water of crystallization, 20 g / L corn steep liquor, 45 g / L CaCO3, and a pH of 6.7.

[0017] This invention provides the use of the culture medium of the composition in the production of 2-ketogluconic acid.

[0018] This invention provides a method for producing 2-ketogluconic acid, comprising the following steps:

[0019] The 2-ketogluconic acid producing bacteria strain was inoculated into the total synthetic medium and cultured under shaking conditions to obtain the proliferated seed liquid;

[0020] The seed culture was inoculated into the fermentation medium and fermented under shaking conditions to obtain 2-ketogluconic acid.

[0021] Preferably, the rotation speed of the proliferation culture or fermentation culture is 250-275 rpm;

[0022] The temperature for the proliferation or fermentation culture is 28–32°C.

[0023] The proliferation culture time is 18–26 hours;

[0024] The fermentation culture time is 64–74 hours.

[0025] The fully synthetic culture medium for the proliferation of 2-ketogluconic acid-producing bacteria provided by this invention comprises the following components per liter: glucose 10.0–40.0 g, urea 0.5–4.0 g, thiamine 5.0–30.0 mg, biotin 0.5–3.0 mg, NaH₂PO₄ 1.0–4.0 g, KH₂PO₄ 0.7–2.1 g, MgSO₄·7H₂O 0.1–0.3 g, and CaCO₃ 0.0–5.0 g, with a pH of 6.0–7.0. This invention uses glucose as the carbon source, urea as the nitrogen source, thiamine and biotin as vitamins, and NaH₂PO₄, KH₂PO₄, MgSO₄·7H₂O, and CaCO₃ as inorganic salts, providing the necessary nutrients for the proliferation of 2-ketogluconic acid-producing bacteria. In this embodiment of the invention, the fully synthetic culture medium and the semi-synthetic culture medium provided by the present invention were used for proliferation culture. The results showed that the bacterial strains in the two culture media were almost identical in glucose utilization, 2-ketogluconic acid synthesis and catabolism, and cell growth. The pH change trends in the later stages of the culture were also basically the same, while the fully synthetic culture medium mitigated the pH changes in the early stages of culture. This may be due to the buffering effect of the fully synthetic culture medium. Therefore, the fully synthetic culture medium provided by the present invention can completely replace the semi-synthetic culture medium without affecting the basic physiological metabolic characteristics and growth and development characteristics of the 2-ketogluconic acid-producing bacteria, thus ensuring the proliferation activity of the 2-ketogluconic acid-producing bacteria.

[0026] Meanwhile, this invention also provides a combined culture medium for the stable production of 2-ketogluconic acid, comprising the aforementioned fully synthetic culture medium and a fermentation culture medium; the fermentation culture medium comprises 160-180 g / L glucose, 18-22 g / L corn steep liquor, 43-47 g / L CaCO3, and a pH of 6.6-6.8. This invention utilizes 2-ketogluconic acid-producing bacteria cultured in the fully synthetic culture medium for fermentation production of 2-ketogluconic acid. Results show that the strains cultured in the fully synthetic and semi-synthetic culture media exhibit almost identical trends in glucose utilization, 2-ketogluconic acid synthesis, cell growth, and pH changes in the fermentation broth. These results indicate that replacing the semi-synthetic culture medium with the fully synthetic culture medium does not affect the catalytic activity of the 2-ketogluconic acid-producing bacteria in catalyzing the oxidation of glucose to 2-ketogluconic acid. Attached Figure Description

[0027] Figure 1 The results of culturing 2-ketogluconic acid-producing bacteria in two different culture media are shown. A represents glucose utilization; B represents 2-ketogluconic acid synthesis and catabolism; C represents bacterial biomass; and D represents culture medium pH.

[0028] Figure 2 The results of fermentation production of 2-ketogluconic acid using strains propagated in two different culture media are shown. A represents glucose utilization; B represents 2-ketogluconic acid synthesis; C represents cell biomass; and D represents the pH of the culture medium.

[0029] Figure 3 The results show the proliferation culture of 2-ketogluconic acid producing bacteria using fully synthetic culture media with or without thiamine and biotin.

[0030] Figure 4 The results show the proliferation culture of 2-ketogluconic acid-producing bacteria using a fully synthetic culture medium containing different vitamins.

[0031] Information on the preservation of biological materials

[0032] Pseudomonas plecoglossicida JSU01 is deposited at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on January 18, 2013, with accession number CGMCC No. 7150. Detailed Implementation

[0033] This invention provides a fully synthetic culture medium for the proliferation of 2-ketogluconic acid-producing bacteria, comprising the following components per liter: glucose 10.0–40.0 g, nitrogen source 0.5–4.0 g, thiamine 5.0–30.0 mg, biotin 0.5–3.0 mg, NaH₂PO₄ 1.0–4.0 g, KH₂PO₄ 0.7–2.1 g, MgSO₄·7H₂O 0.1–0.3 g, CaCO₃ 0.0–5.0 g, with a pH of 6.0–7.0.

[0034] In this invention, each liter of culture medium preferably includes the following components:

[0035] The medium contains 20.0 g / L glucose, 2.0 g / L nitrogen source, 17.5 mg / L thiamine, 1.5 mg / L biotin, 2.1 g / L NaH₂PO₄, 1.4 g / L KH₂PO₄, 0.2 g / L MgSO₄·7H₂O, and 5.0 g / L CaCO₃, with a pH of 6.7. The nitrogen source includes at least one of the following: urea, ammonium chloride, ammonium nitrate, ammonium sulfate, or sodium nitrate. The glucose is preferably glucose containing one molecule of water of crystallization. Compared to semi-synthetic culture media used in industrial production, this invention primarily uses clearly defined components (thiamine, biotin, and NaH₂PO₄) instead of the complex corn steep liquor, while keeping other components unchanged, thus achieving the propagation and culture function of a semi-synthetic culture medium.

[0036] This invention provides the application of the fully synthetic culture medium in the proliferation culture of 2-ketogluconic acid-producing bacteria.

[0037] In this invention, the 2-ketogluconic acid producing bacteria preferably includes at least one of the following microbial strains: *Pseudomonas*, *Erwinia*, *Acetobacter*, *Serratia*, and *Gluconobacter*. The *Pseudomonas* genus preferably includes at least one of the following: *Pseudomonas plecoglossicida*, *Pseudomonas putida*, and *Pseudomonas fluorescens*. In an embodiment of this invention, *Pseudomonas plecoglossicida* JSU01, which has high fermentation yield and a short fermentation cycle, was used as the strain to verify the results of cultivation under different culture media. The results showed that the strains proliferating in the synthetic and semi-synthetic culture media were almost identical in glucose utilization, 2-ketogluconic acid synthesis and catabolism, cell growth, and pH value in the later stages of culture. Furthermore, the buffering components in the synthetic culture medium reduced the drastic pH changes in the early stages of the culture medium. This indicates that fully synthetic culture media can completely replace semi-synthetic culture media without affecting the basic physiological and metabolic characteristics and growth and development characteristics of 2-ketogluconic acid-producing bacteria.

[0038] This invention provides a combined culture medium for the stable production of 2-ketogluconic acid, comprising the total synthetic culture medium and the fermentation culture medium;

[0039] The fermentation medium comprises 160–180 g / L glucose, 18–22 g / L corn steep liquor, 43–47 g / L CaCO3, and a pH of 6.6–6.8.

[0040] In this invention, the fermentation medium preferably comprises 180 g / L glucose containing one molecule of water of crystallization, 20 g / L corn steep liquor, 45 g / L CaCO3, and a pH of 6.7.

[0041] This invention provides the use of the culture medium of the composition in the production of 2-ketogluconic acid.

[0042] This invention provides a method for producing 2-ketogluconic acid, comprising the following steps:

[0043] The 2-ketogluconic acid producing bacteria strain was inoculated into the total synthetic medium and cultured under shaking conditions to obtain the proliferated seed liquid;

[0044] The seed culture was inoculated into the fermentation medium and fermented under shaking conditions to obtain 2-ketogluconic acid.

[0045] In this invention, the rotation speed of the proliferation or fermentation culture is preferably 250–275 rpm, more preferably 265 rpm. The temperature of the proliferation or fermentation culture is preferably 28–32°C, more preferably 30°C. The duration of the proliferation culture is preferably 18–26 h, more preferably 20 h. The duration of the fermentation culture is preferably 64–74 h, more preferably 68 h.

[0046] This invention does not impose any particular limitation on the species of 2-ketogluconic acid-producing bacteria; any species well-known in the art can be used, such as strains of *Pseudomonas*, *Erwinia*, *Acetobacter*, *Serratia*, and *Gluconobacter*. The *Pseudomonas* species preferably include at least one of the following: *Pseudomonas plecoglossicida*, *Pseudomonas putida*, and *Pseudomonas fluorescens*. The inoculum amount of the 2-ketogluconic acid-producing bacteria strain or seed culture is preferably 8%–12%, and most preferably 10%.

[0047] In the embodiments of this invention, the bacterial strains propagated using two different culture media (fully synthetic medium and semi-synthetic medium) showed almost identical trends in glucose utilization, 2-ketogluconic acid synthesis, cell growth, and pH changes in the fermentation broth. This indicates that replacing the semi-synthetic medium with a fully synthetic medium does not affect the activity of the 2-ketogluconic acid-producing bacteria in catalyzing the oxidation of glucose to 2-ketogluconic acid, and can be widely applied in industrial production.

[0048] The following detailed description, in conjunction with embodiments, of a fully synthetic culture medium for the proliferation of 2-ketogluconic acid-producing bacteria provided by the present invention and its application, should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] Proliferation experiments of 2-ketogluconic acid-producing bacteria using different culture media

[0051] 1. Bacterial strain: Pseudomonas plecoglossicida JSU01, obtained by screening and physicochemical mutagenesis from soil.

[0052] 2. Two culture medium formulations

[0053] A commonly used semi-synthetic culture medium in industrial production consists of: 20.0 g / L glucose containing one molecule of water of crystallization, 10.0 g / L corn steep liquor, 2.0 g / L urea, 2.0 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 5.0 g / L light CaCO3, and pH adjusted to 6.7.

[0054] The fully synthetic culture medium consisted of 20.0 g / L glucose, 2.0 g / L urea, 2.1 g / L NaH2PO4, 1.4 g / L KH2PO4, 0.2 g / L MgSO4·7H2O, 17.5 mg / L thiamine, 1.5 mg / L biotin, and 5.0 g / L light CaCO3, with the pH adjusted to 6.7.

[0055] 3. Proliferation and Culture Methods

[0056] The seed culture was performed in shake flasks, and the specific steps were as follows: The JSU01 strain was inoculated into a 500mL Erlenmeyer flask containing 50mL of culture medium (semi-synthetic or fully synthetic medium), and cultured with shaking at 30℃ and 265r / min (25mm offset) for 24h. After the culture, the biomass (OD) of the strain was compared under different culture medium conditions. 650nm Changes in glucose content, 2-ketogluconic acid content, and pH of the culture medium.

[0057] Results of shake-flask seed culture as follows Figure 1 As shown. From Figure 1 As can be seen from the data, the bacterial strains in the two culture media utilized glucose (… Figure 1 Synthesis and catabolism of 2-ketogluconic acid (A), 2-ketogluconic acid Figure 1 (B) and cell growth ( Figure 1 The results in both media were almost identical. The pH trends of the culture solutions were also largely similar in both media. Figure 1 (D), but there are significant differences in the pH variation range in the early stage of culture, which is due to the buffering capacity of the fully synthetic medium.

[0058] The above results indicate that using fully synthetic culture medium instead of semi-synthetic culture medium does not affect the basic physiological and metabolic characteristics and growth and development characteristics of 2-ketogluconic acid-producing bacteria.

[0059] Example 2

[0060] Method for producing 2-ketogluconic acid by fermentation using strains propagated in different culture media as described in Example 1

[0061] Fermentation medium: containing 180.0 g / L glucose with 1 hydrate (162.0 g / L anhydrous glucose), 20.0 g / L corn steep liquor, 45.0 g / L light CaCO3, pH 6.7.

[0062] Shake-flask fermentation: 4 mL of seed culture (the strain propagated on the semi-synthetic or fully synthetic medium prepared in Example 1) was inoculated into a 500 mL Erlenmeyer flask containing 40 mL of fermentation medium and cultured with shaking at 30 °C and 265 r / min (25 mm eccentricity) for 72 h. After the culture was completed, the differences in the fermentation production of 2-ketogluconic acid by the strains propagated on different media were compared.

[0063] The results are as follows Figure 2 As shown. From Figure 2 It can be seen that the bacterial strains proliferated using the two culture media have different glucose utilization rates. Figure 2 Synthesis of A) and 2-ketogluconic acid ( Figure 2 (B) Cell growth (biomass OD) 650nm The formation, Figure 2 The trend of changes in pH value of fermentation broth (C) and other factors. Figure 2 The results from the above studies indicate that replacing the semi-synthetic medium with a fully synthetic medium does not affect the activity of the 2-ketogluconic acid-producing bacteria in catalyzing the oxidation of glucose to 2-ketogluconic acid.

[0064] Example 3

[0065] Proliferation experiments were conducted on 2-ketogluconic acid-producing bacteria using different fully synthetic culture media.

[0066] 1. Bacterial strain: Pseudomonas plecoglossicida JSU01.

[0067] 2. Two formulations of fully synthetic culture media

[0068] Totally synthetic medium 1: glucose 20.0 g / L containing 1 molecule of water of crystallization, urea 2.0 g / L, NaH2PO4 2.1 g / L, KH2PO4 1.4 g / L, MgSO4·7H2O 0.2 g / L, light CaCO3 5.0 g / L, pH adjusted to 6.7.

[0069] Totally synthetic medium 2: glucose 20.0 g / L containing one molecule of water of crystallization, urea 2.0 g / L, NaH2PO4 2.1 g / L, KH2PO4 1.4 g / L, MgSO4·7H2O 0.2 g / L, thiamine 17.5 mg / L, biotin 1.5 mg / L, light CaCO3 5.0 g / L, pH adjusted to 6.7.

[0070] 3. Proliferation and Culture Methods

[0071] Same as Example 1.

[0072] The results of shake-flask seed culture showed that the strains propagated using the two fully synthetic media did not exhibit significant differences in glucose utilization, 2-ketogluconic acid synthesis and catabolism, or pH trends. However, there were differences in cell growth (biomass OD). 650nm The formation, Figure 3 There are obvious differences in the two aspects: (1) The cell biomass of the fully synthetic medium 2 containing thiamine and biotin can reach more than 16.35, which is more than 25% higher than that of the fully synthetic medium 1 without thiamine and biotin; (2) In the fully synthetic medium 1 without thiamine and biotin, the cell lysis phenomenon is very obvious in the later stage of culture (after 18 hours of culture).

[0073] Example 4

[0074] 2-Ketogluconic acid was produced by fermentation using strains propagated in different total synthetic culture media as described in Example 3. The fermentation medium and shake-flask fermentation method were the same as in Example 2.

[0075] Fermentation results showed that when the strain propagated on a fully synthetic medium without thiamine and biotin was used for 2-ketogluconic acid fermentation, the shake-flask fermentation period was 72 h, the fermentation yield was 150.43 g / L, the molar conversion rate of glucose was 85.30%, and the fermentation production intensity was 2.09 g / L / h. When the strain propagated on a fully synthetic medium containing thiamine and biotin was used for 2-ketogluconic acid fermentation, the shake-flask fermentation period was 64 h, the fermentation yield was 160.68 g / L, the molar conversion rate of glucose was 91.11%, and the fermentation production intensity was 2.51 g / L / h.

[0076] Example 5

[0077] A proliferation culture experiment was conducted on 2-ketogluconic acid-producing bacteria using a fully synthetic culture medium containing different vitamins.

[0078] 1. Bacterial strain: Pseudomonas plecoglossicida JSU01.

[0079] 2. Two formulations of fully synthetic culture media

[0080] Totally synthetic culture medium 1: glucose 20.0 g / L containing one molecule of water of crystallization, urea 2.0 g / L, NaH2PO4 2.1 g / L, KH2PO4 1.4 g / L, MgSO4·7H2O 0.2 g / L, vitamin C 17.5 mg / L, vitamin B2 1.5 mg / L, light CaCO3 5.0 g / L, pH adjusted to 6.7.

[0081] Totally synthetic medium 2: glucose 20.0 g / L containing one molecule of water of crystallization, urea 2.0 g / L, NaH2PO4 2.1 g / L, KH2PO4 1.4 g / L, MgSO4·7H2O 0.2 g / L, thiamine 17.5 mg / L, biotin 1.5 mg / L, light CaCO3 5.0 g / L, pH adjusted to 6.7.

[0082] 3. Proliferation and Culture Methods

[0083] Same as Example 1.

[0084] The results of shake-flask seed culture showed that the strains propagated using the two fully synthetic media did not exhibit significant differences in glucose utilization, 2-ketogluconic acid synthesis and catabolism, or pH trends. However, there were differences in cell growth (biomass OD). 650nm The formation, Figure 4 There are significant differences, mainly in two aspects: (1) The total synthetic medium containing thiamine and biotin increases the cell biomass (OD) of the proliferating cells. 650nm (1) The concentration can reach more than 16.40, which is more than 25% higher than that of the total synthetic medium 1 containing vitamin C and vitamin B2; (2) In the total synthetic medium 1, the cell lysis phenomenon is very obvious in the later stage of culture (after 18 hours of culture).

[0085] Example 6

[0086] 2-Ketogluconic acid was produced by fermentation using strains propagated in different total synthetic culture media as described in Example 5. The fermentation medium and shake-flask fermentation method were the same as in Example 2.

[0087] Fermentation results showed that when the strain propagated on a fully synthetic medium containing vitamin C and vitamin B2 was used for 2-ketogluconic acid fermentation, the shake-flask fermentation period was 72 h, the fermentation yield was 152.64 g / L, the molar conversion rate of glucose was 86.55%, and the fermentation production intensity was 2.12 g / L / h. When the strain propagated on a fully synthetic medium containing thiamine and biotin was used for 2-ketogluconic acid fermentation, the shake-flask fermentation period was 64 h, the fermentation yield was 161.04 g / L, the molar conversion rate of glucose was 91.31%, and the fermentation production intensity was 2.52 g / L / h.

[0088] Example 7

[0089] Different 2-ketogluconic acid producing bacteria were propagated using different culture media, and the propagated strains were used for 2-ketogluconic acid fermentation.

[0090] 1. Bacterial strains: *Pseudomonas proteus* JSU01, *Serratia marcescens* GIM1.216, *Pseudomonas putida* GIM1.203, and *Pseudomonas fluorescens* GIM1.209. Among them, *Serratia marcescens* GIM1.216, *Pseudomonas putida* GIM1.203, and *Pseudomonas fluorescens* GIM1.209 were obtained from the China General Microbiological Culture Collection Center.

[0091] 2. Two culture medium formulations for bacterial proliferation

[0092] Semi-synthetic culture medium: 20.0 g / L glucose with 1 molecule of water of crystallization, 10.0 g / L corn steep liquor, 2.0 g / L urea, 2.0 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 5.0 g / L light CaCO3, pH adjusted to 6.7.

[0093] The fully synthetic culture medium consisted of 20.0 g / L glucose with one molecule of water of crystallization, 1.5 g / L urea, 1.0 g / L NaH2PO4, 1.0 g / L KH2PO4, 0.1 g / L MgSO4·7H2O, 25.0 mg / L thiamine, 2.0 mg / L biotin, and 5.0 g / L light CaCO3, with the pH adjusted to 6.7.

[0094] 3. The fermentation medium and shake-flask fermentation method are the same as in Example 2.

[0095] The results of shake-flask seed culture showed that the various bacterial species in the two proliferation media were almost identical in glucose utilization, 2-ketogluconic acid synthesis and catabolism, and cell growth, and the pH trends of the culture media were also basically similar. At 20 h of culture, the OD of the *Pseudomonas proteus* JSU01 culture medium... 650nm The OD values ​​of the culture media were 16.21 (semi-synthetic medium) and 16.34 (fully synthetic medium), respectively, and the pH values ​​were 7.98 (semi-synthetic medium) and 8.05 (fully synthetic medium), respectively. The OD values ​​of the *Serratia marcescens* GIM1.216 culture medium were... 650nm The OD values ​​of the culture medium were 15.47 (semi-synthetic medium) and 15.26 (fully synthetic medium), respectively, and the pH values ​​were 7.83 (semi-synthetic medium) and 7.76 (fully synthetic medium), respectively. The OD values ​​of *Pseudomonas putida* GIM1.203 culture medium were... 650nmThe OD values ​​of the culture medium were 16.40 (semi-synthetic medium) and 16.29 (fully synthetic medium), respectively, and the pH values ​​were 8.02 (semi-synthetic medium) and 8.07 (fully synthetic medium), respectively. The OD values ​​of the *Pseudomonas fluorescens* GIM1.209 culture medium were... 650nm The pH values ​​were 15.98 (semi-synthetic medium) and 16.09 (fully synthetic medium), respectively, and the pH values ​​were 7.89 (semi-synthetic medium) and 8.01 (fully synthetic medium), respectively.

[0096] The results of shake-flask fermentation showed that the various strains proliferated using the two culture media exhibited differences in glucose utilization, 2-ketogluconic acid synthesis, and cell growth (biomass OD). 650nm The formation of the bacteria and the trend of pH changes in the fermentation broth were almost identical. After 64 h of cultivation, the yield of 2-ketogluconic acid in the fermentation broth of *Pseudomonas proteus* JSU01 was 158.94 g / L (strain propagated in semi-synthetic medium) and 160.10 g / L (strain propagated in fully synthetic medium), respectively; the yield of 2-ketogluconic acid in the fermentation broth of *Serratia marcescens* GIM1.216 was 142.66 g / L (strain propagated in semi-synthetic medium) and 144.05 g / L (strain propagated in fully synthetic medium), respectively. The yields of 2-ketogluconic acid in the fermentation broth of *Pseudomonas putida* GIM1.203 were 144.32 g / L (strain propagated in semi-synthetic medium) and 143.98 g / L (strain propagated in fully synthetic medium), respectively; the yields of 2-ketogluconic acid in the fermentation broth of *Pseudomonas fluorescens* GIM1.209 were 147.85 g / L (strain propagated in semi-synthetic medium) and 146.30 g / L (strain propagated in fully synthetic medium), respectively.

[0097] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fully synthetic culture medium for the proliferation of 2-ketogluconic acid-producing bacteria, characterized in that, Each liter of culture medium consists of the following components: The following are the ingredients: glucose with 1 molecule of water of crystallization (20.0 g / L), urea (2.0 g / L), thiamine (17.5 mg / L), biotin (1.5 mg / L), NaH2PO4 (2.1 g / L), KH2PO4 (1.4 g / L), MgSO4·7H2O (0.2 g / L), CaCO3 (5.0 g / L), and pH 6.

7.

2. The application of the fully synthetic culture medium according to claim 1 in the proliferation culture of 2-ketogluconic acid producing bacteria.

3. The application according to claim 2, characterized in that, The 2-ketogluconic acid-producing bacteria include at least one of the following microbial strains: *Pseudomonas* genus ( Pseudomonas Erwinia ( ) Erwinia Acetic acid bacteria ( Acetobacter ), Serratia ( Serratia ) and Gluconobacterium spp. Gluconobacter ).

4. The application according to claim 3, characterized in that, The *Pseudomonas* genus includes at least one of the following: *Pseudomonas proteus* (… P. plecoglossicida ), Pseudomonas putida ( P. putida ) and fluorescent pseudomonas ( P. fluorescens ).

5. A combined culture medium for the stable production of 2-ketogluconic acid, characterized in that, Includes the fully synthetic culture medium and fermentation culture medium as described in claim 1; The fermentation medium comprises 160-180 g / L glucose with 1 hydrate, 18-22 g / L corn steep liquor, 43-47 g / L CaCO3, and a pH of 6.6-6.

8.

6. The combined culture medium for the stable production of 2-ketogluconic acid according to claim 5, characterized in that, The fermentation medium comprises 180 g / L glucose containing one molecule of water of crystallization, 20 g / L corn steep liquor, 45 g / L CaCO3, and a pH of 6.

7.

7. The use of the combined culture medium of claim 5 or 6 in the production of 2-ketogluconic acid.

8. A method for producing 2-ketogluconic acid, characterized in that, Includes the following steps: The 2-ketogluconic acid producing bacteria strain was inoculated into the fully synthetic culture medium described in claim 1 and cultured under shaking conditions to obtain the proliferated seed liquid. The seed culture was inoculated into the fermentation medium and fermented under shaking conditions to obtain 2-ketogluconic acid.

9. The method according to claim 8, characterized in that, The rotation speed of the propagation or fermentation culture is 250~275 rpm; The temperature for the proliferation or fermentation culture is 28~32℃; The proliferation culture time is 18-26 h; The fermentation culture time is 64-74 h.

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