Application of iminodisuccinate in high-density fermentation of microorganisms

Abstract

The invention provides application of iminodisuccinate in high-density fermentation of microorganisms. The iminodisuccinate comprises at least one of copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate and manganese iminodisuccinate. In a fermentation culture medium, iminodisuccinate is used as an organic trace element to replace an inorganic trace element in the fermentation culture medium. In the high-density fermentation process of Escherichia coli, iminodisuccinate is easier to absorb and utilize by microorganisms than normal inorganic salt, the fermentation biomass can be increased when the iminodisuccinate is used for fermentation within the range of the invention, and fermentation production is more facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to the application of iminodisuccinate in high-density microbial fermentation. Background Art

[0002] High-density fermentation of microorganisms generally refers to the cultivation technology of the growth state of microorganisms in liquid culture when the cell population density exceeds that of conventional culture by more than 10 times. Its application range is now becoming more and more extensive, such as insulin, peptide drugs, enzymes, etc.

[0003] Modern high-density fermentation of E. coli is generally achieved through genetically engineered bacteria. In the process of high-density fermentation, the main influencing factors include culture medium components, fermentation conditions, feeding strategies, etc. The main components of the culture medium ratio are: carbon source, nitrogen source, trace elements, growth factors, etc. Trace elements are divided into metal trace elements and non-metal trace elements, among which metal trace elements mainly include iron, manganese, zinc, copper, calcium, etc. Although their dosage is very small, they still play an important role in the fermentation process. For example, iron is a component of cytochrome enzymes and cytochrome oxidases, zinc is an activator of saccharifying enzymes, copper is an activator of enzymes related to the alcohol oxidation process, and calcium is an activator of proteases. It can regulate cell osmotic pressure and buffer the pH value of the culture medium, etc.

[0004] In fermentation culture, general metal trace elements are added to the culture medium in the form of inorganic salts, such as copper sulfate, zinc sulfate, calcium chloride, etc. In the high-density fermentation process of microorganisms, the absorption rate of inorganic salts is slow. Due to the osmotic pressure inside and outside the cells and the characteristics of the cell membrane itself, sometimes a higher ion concentration is required to promote microbial absorption, but a higher ion concentration may inhibit microbial growth, so the ratio of metal trace elements is a relatively important factor in the fermentation process. Organic salts are absorbed and utilized faster by microorganisms, but organic salts are rarely used for microbial fermentation culture. This is because there are three difficulties in using organic salts for fermentation: First, some organic salts are insoluble in water and are difficult to use in fermentation, such as stearates; second, some organic salts are difficult to prepare and have high prices, which are not conducive to large-scale fermentation, such as amino acid chelates, polypeptide salts, etc.; third, some organic salts cannot be used by microbial fermentation, and even harm the growth of microorganisms, such as EDTA salts. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an application of iminodisuccinate in microbial high-density fermentation, specifically using iminodisuccinate to replace inorganic trace elements to provide trace elements for microbial high-density fermentation. As an organic salt that is easily soluble in water, iminodisuccinate has a simple preparation method and can be absorbed and utilized by microorganisms. In the fermentation medium, iminodisuccinate is used as an organic trace element to replace the inorganic trace elements in the fermentation medium, which can not only solve the problem of absorption and utilization of metal inorganic salt ions during microbial high-density fermentation, but also provide part of the carbon source and nitrogen source for Escherichia coli fermentation.

[0006] The first object of the present invention is to provide an application of iminodisuccinate in high-density fermentation of microorganisms; the iminodisuccinate comprises at least one of copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate and manganese iminodisuccinate.

[0007] Preferably, the preparation method of the iminodisuccinate is: adding maleic anhydride and L-aspartic acid to water and ammonia solution, then adding a metal hydroxide and stirring, heating to 75-95° C. after complete dissolution, heating under reflux reaction at normal pressure, and drying the solution after the reaction is completed to obtain an iminodisuccinate solid; the metal hydroxide includes copper hydroxide, ferrous hydroxide, calcium hydroxide, zinc hydroxide and manganese hydroxide.

[0008] Preferably, the microorganism is Escherichia coli.

[0009] Preferably, in high-density microbial fermentation, the iminodisuccinate is used as a trace element in the microbial fermentation medium.

[0010] Preferably, in the microbial fermentation medium, the concentration of the copper element is 0.0130-0.0280 g / L; and / or

[0011] The concentration of the ferrous element is 0.0210-0.0440 g / L; and / or

[0012] The concentration of the calcium element is 0.0060-0.0170 g / L; and / or

[0013] The concentration of the zinc element is 0.0150-0.0260 g / L; and / or

[0014] The concentration of the manganese element is 0.0012-0.0028 g / L.

[0015] Preferably, in the microbial fermentation medium, the concentration of the copper element is 0.0153-0.0255 g / L; the concentration of the ferrous element is 0.0241-0.0402 g / L; the concentration of the calcium element is 0.0072-0.0144 g / L; the concentration of the zinc element is 0.0182-0.0227 g / L; and the concentration of the manganese element is 0.0015-0.0025 g / L.

[0016] Preferably, in the microbial fermentation medium, the concentration of the copper element is 0.0204 g / L; the concentration of the ferrous element is 0.0321 g / L; the concentration of the calcium element is 0.0108 g / L; the concentration of the zinc element is 0.0205 g / L; and the concentration of the manganese element is 0.0020 g / L.

[0017] The second object of the present invention is a fermentation medium for high-density fermentation of microorganisms, wherein the fermentation medium comprises a trace element mother solution, and the formula of the trace element mother solution is: copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate, and manganese iminodisuccinate;

[0018] In the microbial fermentation medium, the concentration of the copper element is 0.0153-0.0255 g / L; the concentration of the ferrous element is 0.0241-0.0402 g / L; the concentration of the calcium element is 0.0072-0.0144 g / L; the concentration of the zinc element is 0.0182-0.0227 g / L; and the concentration of the manganese element is 0.0015-0.0025 g / L.

[0019] Preferably, in the microbial fermentation medium, the concentration of the copper element is 0.0204 g / L; the concentration of the ferrous element is 0.0321 g / L; the concentration of the calcium element is 0.0108 g / L; the concentration of the zinc element is 0.0205 g / L; and the concentration of the manganese element is 0.0020 g / L.

[0020] The third object of the present invention is to provide a microbial high-density fermentation culture method, comprising the following steps:

[0021] (1) activating the microbial strains and performing primary strain culture and secondary seed culture; preferably, the microorganism is Escherichia coli;

[0022] (2) Fermentation culture: The secondary seeds are inoculated into a fermentation tank at an inoculation rate of 2%-8%, and the fermentation medium is used for fermentation. The rotation speed is coupled with the ventilation volume and dissolved oxygen to control the dissolved oxygen to ≥ 40% of the initial oxygen content, and the pH is controlled to be 6.8-7.2; after the dissolved oxygen rebounds, feed is started, and fermentation is carried out until the fermentation liquid OD 600No more growth, fermentation ends;

[0023] Preferably, the feed medium during feeding is: 300-600 g / L glucose, 8-16 g / L peptone, 8-16 g / L yeast powder, and 3-10 g / L magnesium sulfate heptahydrate.

[0024] In the fermentation medium, iminodisuccinate is used as an organic trace element to replace the inorganic trace elements in the fermentation medium. In the high-density fermentation process of Escherichia coli, iminodisuccinate is more easily absorbed and utilized by microorganisms than normal inorganic salts. Within the scope of the present invention, the use of iminodisuccinate for fermentation can increase the fermentation biomass and is more conducive to fermentation production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0026] Figure 1 OD of each group of Comparative Examples 1-3 and Examples 1-4 600 Comparison chart with fermentation time.

[0027] Figure 2 The fermentation medium prepared in Example 1 and Comparative Example 1. Figure a is the fermentation medium prepared in Example 1, and Figure b is the fermentation medium prepared in Comparative Example 1.

[0028] Figure 3 This is a shake flask culture diagram of Example 1 of the present invention.

[0029] Figure 4 This is a fermentation culture diagram of Example 1 of the present invention.

[0030] Figure 5 It is a comparison diagram of the fermentation broth of Example 1 and Comparative Example 1. Among them, Figure a is the fermentation broth of Example 1, and Figure b is the fermentation broth of Comparative Example 1. DETAILED DESCRIPTION

[0031] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0032] The iminodisuccinates of the present invention include copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate and manganese iminodisuccinate.

[0033] The preparation method of iminodisuccinate of the present invention is as follows:

[0034] 0.8-1.2 molar parts of maleic anhydride and 0.8-1.2 molar parts of L-aspartic acid are added to 15-25 molar parts of water, and then a 25% ammonia solution is added, the amount of ammonia added is 0.4%-0.8% of the mass fraction of water, and then 0.8-1.2 molar parts of the corresponding metal hydroxide (copper hydroxide, ferrous hydroxide, calcium hydroxide, zinc hydroxide, manganese hydroxide) are slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 75-95° C., heated under normal pressure for reflux reaction, cooled to room temperature after the reaction is completed, and dried to obtain the corresponding iminodisuccinate solid (iminodisuccinate copper, iminodisuccinate ferrous, iminodisuccinate calcium, iminodisuccinate zinc and iminodisuccinate manganese). In the iminodisuccinate, the content of iminodisuccinate ion is 70-80%, and the content of salt is 15-20%.

[0035] When preparing iminodisuccinate, ammonia water must be used as an auxiliary agent, and other auxiliary agents may affect the fermentation of Escherichia coli.

[0036] When the iminodisuccinate of the present invention is applied to high-density microbial fermentation, it is used as a trace element in the microbial fermentation medium.

[0037] When used in a microbial fermentation medium, the trace element includes one or more of copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate and manganese iminodisuccinate;

[0038] Wherein, in the microbial fermentation medium, the concentration of the copper element is 0.0130-0.0280 g / L; and / or

[0039] The concentration of the ferrous element is 0.0210-0.0440 g / L; and / or

[0040] The concentration of the calcium element is 0.0060-0.0170 g / L; and / or

[0041] The concentration of the zinc element is 0.0150-0.0260 g / L; and / or

[0042] The concentration of the manganese element is 0.0012-0.0028 g / L.

[0043] When the culture medium is prepared within the above range and fermented with microorganisms, a higher biomass can be obtained.

[0044] The present invention only limits the trace element components in the microbial fermentation medium, and has no specific requirements for other components in the medium, as long as high-density fermentation of microorganisms can be achieved.

[0045] For example, when the microorganism is Escherichia coli, the formula of the Escherichia coli fermentation medium can be: glucose 5-50g / L, peptone 2-20g / L, yeast powder 2-20g / L, potassium dihydrogen phosphate 1-5g / L, diammonium hydrogen phosphate 10-30g / L, sodium citrate monohydrate 1-5g, trace element mother solution 20mL / L, defoamer 0.1-1g / L;

[0046] The formula of the trace element mother solution is: copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate and manganese iminodisuccinate.

[0047] Wherein, in the Escherichia coli fermentation medium, the concentration of the copper element is 0.0153-0.0255 g / L; for example, it can be: 0.0153 g / L, 0.0163 g / L, 0.0173 g / L, 0.0190 g / L, 0.0210 g / L, 0.0233 g / L, 0.0255 g / L.

[0048] The concentration of the ferrous element is 0.0241-0.0402 g / L; for example, it can be: 0.0241 g / L, 0.0261 g / L, 0.0281 g / L, 0.0301 g / L, 0.0341 g / L, 0.0381 g / L, 0.0402 g / L.

[0049] The concentration of the calcium element is 0.0072-0.0144 g / L; for example, it can be: 0.0072 g / L, 0.0082 g / L, 0.0092 g / L, 0.0102 g / L, 0.0112 g / L, 0.0122 g / L, 0.0132 g / L, 0.0144 g / L.

[0050] The concentration of the zinc element is 0.0182-0.0227 g / L; for example, it can be: 0.0182 g / L, 0.0192 g / L, 0.0197 g / L, 0.0202 g / L, 0.0207 g / L, 0.0212 g / L, 0.0217 g / L, 0.0222 g / L, 0.0227 g / L.

[0051] The concentration of the manganese element is 0.0015-0.0025 g / L; for example, it can be: 0.0015 g / L, 0.0017 g / L, 0.0019 g / L, 0.0021 g / L, 0.0023 g / L, 0.0025 g / L.

[0052] The trace element mother solution is prepared by dissolving the above components in water in proportion and fixing the volume to 1L.

[0053] When the culture medium is prepared within the above range and fermented with microorganisms, a higher biomass can be obtained.

[0054] In the most preferred embodiment of the present invention, the formula of the Escherichia coli fermentation medium is: glucose 25g / L, peptone 14.5g / L, yeast powder 10.5g / L, potassium dihydrogen phosphate 2g / L, diammonium hydrogen phosphate 14g / L, sodium citrate monohydrate 3.5g, trace element mother solution 20mL / L, defoamer 0.2g / L;

[0055] The formula of the trace element mother solution is: copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate and manganese iminodisuccinate.

[0056] Among them, in the Escherichia coli fermentation medium, the concentration of copper element is 0.0204g / L; the concentration of ferrous element is 0.0321g / L; the concentration of calcium element is 0.0108g / L; the concentration of zinc element is 0.0205g / L; and the concentration of manganese element is 0.0020g / L.

[0057] At this time, the final biomass after E. coli fermentation was the highest.

[0058] The method for high-density fermentation of Escherichia coli using the Escherichia coli fermentation medium of the present invention is as follows:

[0059] 1. Activate the Escherichia coli strains and carry out primary strain culture and secondary seed culture;

[0060] 2. Fermentation culture

[0061] The secondary seeds were inoculated into the fermentation tank at a rate of 2%-8%, with an initial speed of 200rpm and an initial ventilation ratio of 1.5:1. The speed was coupled with the ventilation volume and dissolved oxygen to control the dissolved oxygen to ≥ 40% of the initial oxygen content. The pH was controlled at 6.8-7.2 by adding ammonia water. Feeding was started after the dissolved oxygen rebounded, and the feed rate was adjusted to control the residual sugar concentration at 1.0-1.5g / L. The fermentation was continued until the OD value of the fermentation liquid reached 0.1%. 600 The fermentation medium is the fermentation medium of the present invention; the feed medium formula is: glucose 300-600g / L, peptone 8-16g / L, yeast powder 8-16g / L, magnesium sulfate heptahydrate 3-10g / L.

[0062] The activation of the Escherichia coli strains is specifically as follows:

[0063] On a clean bench, use an inoculation loop to take an appropriate amount of bacteria from the glycerol tube of the bacteria and streak on a plate culture medium, and culture at 35-39°C for 12-16 hours to obtain activated bacteria. The plate culture medium is solid LB medium or solid TB medium.

[0064] The specific primary bacterial culture is as follows:

[0065] On the clean bench, pick a single colony from the plate with an inoculation loop and add it to the test tube culture medium. Cultivate at 35-39°C and 150-250 rpm until OD 600 ≥ 1. The test tube culture medium is liquid LB medium or liquid TB medium.

[0066] Secondary seed cultivation is as follows:

[0067] Inoculate the primary strain into the shake flask medium at a rate of 1%-2% v / v on a clean bench and culture at 35-39°C and 150-250 rpm until OD 600 ≥ 2. The shake flask medium is liquid LB medium or liquid TB medium.

[0068] Comparative Example 1 High-density fermentation culture of Escherichia coli

[0069] 1. Activation of E. coli strains: Place the plate culture medium in a conical flask, seal it, and place it in an autoclave. Sterilize it at 121°C and 0.1Mpa for 20 minutes. When the temperature drops to 50°C, pour the plate. After the plate culture medium is completely cooled and solidified, use an inoculation loop to dip a small amount of E. coli cells from the glycerol tube on the clean bench and streak them on the plate culture medium in three stages. Incubate at 37°C for 12 hours to obtain activated strains.

[0070] Among them, the plate culture medium is solid LB culture medium with the formula: 10g / L tryptone, 5g / L yeast powder, 10g / L sodium chloride, and 20g / L agar powder.

[0071] 2. Primary bacterial culture: Put the liquid culture medium into a test tube, seal it, and sterilize it in an autoclave at 121°C and 0.1Mpa for 20 minutes. After cooling, pick a single colony from the plate culture medium on a clean bench and put it into the test tube culture medium. Cultivate it at 37°C and 200rpm until the OD reaches 600 ≥1.

[0072] Among them, the test tube culture medium is liquid LB culture medium with the formula of: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L.

[0073] 3. Secondary seed culture: put the liquid culture medium into the shake flask with a volume of 30mL / 250mL, seal it, and sterilize it in the autoclave at 121℃ and 0.1Mpa for 20min. After cooling, inoculate the primary strain into the shake flask culture medium at a 2% inoculation rate on the clean bench, and culture it at 37℃ and 200rpm until the OD 600 ≥2.

[0074] The shake flask culture medium is liquid LB culture medium with the following formula: 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L sodium chloride.

[0075] 4. Fermentation culture: Prepare 2L fermentation medium and put it into a 5L fermentation tank, seal it well, sterilize it at 121℃, 0.1Mpa for 20min, and set the dissolved oxygen of the solution to 100%. Prepare 1.5L feed medium and put it into the feed bottle, seal it well, and sterilize it at 121℃, 0.1Mpa for 20min.

[0076] After the culture medium has cooled, use flame inoculation to inoculate the secondary seeds into the fermentation medium at a 6% inoculation rate. Set the temperature to 37°C, the initial speed to 200rpm, the initial ventilation ratio to 1.5:1, start stirring, couple the stirring speed with the ventilation volume and the dissolved oxygen in the fermentation liquid, set the dissolved oxygen ≥ 40% of the initial oxygen content, and control the pH of the fermentation liquid to 6.8-7.2 by feeding with ammonia water to start fermentation. During fermentation, as E. coli multiplies, the dissolved oxygen in the solution will gradually decrease. When it decreases to a certain level, the nutrients in the solution are consumed, and the microorganisms no longer grow and consume oxygen, resulting in a rebound in dissolved oxygen. At this time, start feeding, take manual samples every hour, and detect the residual sugar content and OD 600 , adjust the feeding rate, and control the residual sugar content to 1.0-1.5g / L. If the foam is too high during the fermentation process, add defoamer. 600 The tank is lowered without raising the temperature any more to obtain a high-density Escherichia coli fermentation liquid.

[0077] Among them, the fermentation medium formula is: glucose 25g / L, peptone 14.5g / L, yeast powder 10.5g / L, potassium dihydrogen phosphate 2g / L, diammonium hydrogen phosphate 14g / L, sodium citrate monohydrate 3.5g, trace element mother solution 20mL / L, defoamer 0.2g / L.

[0078] The formula of the trace element mother solution is: 4 g / L of copper sulfate pentahydrate, 8 g / L of ferrous sulfate heptahydrate, 1.5 g / L of calcium chloride, 4.5 g / L of zinc sulfate heptahydrate, and 0.4 g / L of manganese sulfate tetrahydrate.

[0079] The preparation method of the trace element mother solution is: dissolve the above components in water according to proportion and make the volume to 1L.

[0080] The feed medium formula is: peptone 10g / L, yeast powder 10g / L, magnesium sulfate heptahydrate 6g / L, glucose 400g / L.

[0081] Example 1 High-density fermentation culture of Escherichia coli

[0082] Compared with Comparative Example 1, the present embodiment is different in that: the formula of the trace element mother solution is: 5.69 g / L of copper iminodisuccinate, 8.95 g / L of ferrous iminodisuccinate, 3 g / L of calcium iminodisuccinate, 5.64 g / L of zinc iminodisuccinate, and 0.55 g / L of manganese iminodisuccinate (converted according to the trace element content in Comparative Example 1).

[0083] The preparation method of the trace element mother solution is: dissolve the above components in water according to proportion and make the volume to 1L.

[0084] The remaining parameters and preparation methods are the same as those in Comparative Example 1.

[0085] Wherein, the preparation method of iminodisuccinate is as follows:

[0086] Copper iminodisuccinate:

[0087] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.5% of the mass fraction of water, and then 1.0 mole of copper hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 80° C., heated under reflux at normal pressure for 25 hours, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain iminodisuccinate copper solid. The mass percentage of iminodisuccinate in the solid is 75%, and the mass percentage of copper is 18%.

[0088] Ferrous iminodisuccinate:

[0089] 0.8 molar parts of maleic anhydride and 0.8 molar parts of L-aspartic acid are added to 20 molar parts of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.6% of the mass fraction of water, and then 1.1 molar parts of ferrous hydroxide are slowly added and stirred, and the raw materials are heated to 80° C. after being completely dissolved, and heated under reflux for 32 hours at normal pressure. After the reaction is completed, the temperature is cooled to room temperature, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain ferrous iminodisuccinate solid. The mass percentage of iminodisuccinate in the solid is 79%, and the mass percentage of ferrous iron is 18%.

[0090] Calcium iminodisuccinate:

[0091] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.8% of the mass fraction of water, and then 0.9 mole of calcium hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 80°C, heated under reflux at normal pressure for 28 hours, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120°C. The mass percentage of iminodisuccinate in the solid is 77%, and the mass percentage of calcium is 18%.

[0092] Zinc iminodisuccinate:

[0093] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.4% of the mass fraction of water, and then 1.1 mole of zinc hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, the mixture is heated to 80° C., and heated under reflux for reaction for 25 hours at normal pressure. After the reaction is completed, the mixture is cooled to room temperature, insoluble matter is filtered, and the solution is dried at 120° C. to obtain solid zinc iminodisuccinate. The mass percentage of iminodisuccinate in the solid is 77%, and the mass percentage of zinc is 18%.

[0094] Manganese iminodisuccinate:

[0095] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.4% of the mass fraction of water, and then 1.0 mole of manganese hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 80° C., heated under reflux at normal pressure for 25 hours, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain iminodisuccinate manganese solid. The mass percentage of iminodisuccinate in the solid is 75%, and the mass percentage of manganese is 18%.

[0096] Comparative Example 2 High-density fermentation culture of Escherichia coli

[0097] The difference between this comparative example and comparative example 1 is that: 3 g / L of copper sulfate pentahydrate, 6 g / L of ferrous sulfate heptahydrate, 1.0 g / L of calcium chloride, 4 g / L of zinc sulfate heptahydrate, and 0.3 g / L of manganese sulfate tetrahydrate.

[0098] The remaining parameters and preparation methods are the same as those in Comparative Example 1.

[0099] Example 2 High-density fermentation culture of Escherichia coli

[0100] Compared with Example 1, the difference between this embodiment and Example 1 is that the formula of the trace element mother solution is: 4.27 g / L of copper iminodisuccinate, 6.71 g / L of ferrous iminodisuccinate, 2.00 g / L of calcium iminodisuccinate, 5.02 g / L of zinc iminodisuccinate, and 0.41 g / L of manganese iminodisuccinate (converted according to the trace element content in Comparative Example 2).

[0101] The remaining parameters and preparation methods are the same as those in Example 1.

[0102] Comparative Example 3 High-density fermentation culture of Escherichia coli

[0103] The difference between this comparative example and comparative example 1 is that: 5 g / L of copper sulfate pentahydrate, 10 g / L of ferrous sulfate heptahydrate, 2 g / L of calcium chloride, 5 g / L of zinc sulfate heptahydrate, and 0.5 g / L of manganese sulfate tetrahydrate.

[0104] The remaining parameters and preparation methods are the same as those in Comparative Example 1.

[0105] Example 3 High-density fermentation culture of Escherichia coli

[0106] Compared with Example 1, the difference between this embodiment and Example 1 is that the formula of the trace element mother solution is: 7.11 g / L of copper iminodisuccinate, 11.19 g / L of ferrous iminodisuccinate, 4 g / L of calcium iminodisuccinate, 6.27 g / L of zinc iminodisuccinate, and 0.69 g / L of manganese iminodisuccinate (converted according to the trace element content in Comparative Example 3).

[0107] The remaining parameters and preparation methods are the same as those in Example 1.

[0108] Example 4 High-density fermentation culture of Escherichia coli

[0109] Compared with Example 1, the difference between this embodiment and Example 1 is that the formula of the trace element mother solution is: 5.12 g / L of copper iminodisuccinate, 8.06 g / L of ferrous iminodisuccinate, 2.7 g / L of calcium iminodisuccinate, 5.08 g / L of zinc iminodisuccinate, and 0.40 g / L of manganese iminodisuccinate (the trace element content is reduced by 10% compared with Example 1).

[0110] The remaining parameters and preparation methods are the same as those in Example 1.

[0111] Example 5 High-density fermentation culture of Escherichia coli

[0112] Compared with Example 1, this embodiment is different in that:

[0113] The preparation method of iminodisuccinates is as follows:

[0114] Copper iminodisuccinate:

[0115] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.4% of the mass fraction of water, and then 0.8 mole of copper hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 80° C., heated under reflux for 25 hours at normal pressure, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain iminodisuccinate copper solid. The mass percentage of iminodisuccinate in the solid is 77%, and the mass percentage of copper is 15%.

[0116] Ferrous iminodisuccinate:

[0117] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.5% of the mass fraction of water, and then 0.9 mole of ferrous hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 80° C., heated under reflux for 30 hours at normal pressure, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain ferrous iminodisuccinate solid. The mass percentage of iminodisuccinate in the solid is 78%, and the mass percentage of ferrous iron is 15%.

[0118] Calcium iminodisuccinate:

[0119] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then a 25% ammonia solution is added, the amount of ammonia added is 0.6% of the mass fraction of water, and then 0.8 mole of calcium hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 80°C, heated under reflux at normal pressure for 25 hours, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120°C. The mass percentage of iminodisuccinate in the solid is 78%, and the mass percentage of calcium is 15%.

[0120] Zinc iminodisuccinate:

[0121] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.4% of the mass fraction of water, and then 0.9 mole of zinc hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, the mixture is heated to 80° C., and heated under reflux for 25 hours at normal pressure. After the reaction is completed, the mixture is cooled to room temperature, insoluble matter is filtered, and the solution is dried at 120° C. to obtain solid zinc iminodisuccinate. The mass percentage of iminodisuccinate in the solid is 77%, and the mass percentage of zinc is 18%.

[0122] Manganese iminodisuccinate:

[0123] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.4% of the mass fraction of water, and then 0.8 mole of manganese hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 80° C., heated under reflux at normal pressure for 22 hours, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain iminodisuccinate manganese solid. The mass percentage of iminodisuccinate in the solid is 79%, and the mass percentage of manganese is 15%.

[0124] The remaining parameters and preparation methods are the same as those in Example 1.

[0125] In this example, only the concentration of the prepared iminodisuccinate is slightly different from that in Example 1. It can be predicted that the effect of the fermentation medium prepared by using the same on the high-density fermentation culture of Escherichia coli is also similar to that in Example 1.

[0126] Example 6 High-density fermentation culture of Escherichia coli

[0127] Compared with Example 1, this embodiment is different in that:

[0128] The preparation method of iminodisuccinates is as follows:

[0129] Copper iminodisuccinate:

[0130] 0.9 molar parts of maleic anhydride and 0.9 molar parts of L-aspartic acid are added to 20 molar parts of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.7% of the mass fraction of water, and then 1.2 molar parts of copper hydroxide are slowly added and stirred. After the raw materials are completely dissolved, they are heated to 88° C., heated under reflux for 30 hours at normal pressure, and cooled to room temperature after the reaction is completed. Insoluble matter is filtered, and the solution is dried at 120° C. to obtain iminodisuccinate copper solid. The mass percentage of iminodisuccinate in the solid is 75%, and the mass percentage of copper is 20%.

[0131] Ferrous iminodisuccinate:

[0132] 0.8 molar parts of maleic anhydride and 0.8 molar parts of L-aspartic acid are added to 20 molar parts of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.8% of the mass fraction of water, and then 1.2 molar parts of ferrous hydroxide are slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 90° C., heated under reflux reaction at normal pressure for 35 hours, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain ferrous iminodisuccinate solid. The mass percentage of iminodisuccinate in the solid is 76%, and the mass percentage of ferrous iron is 20%.

[0133] Calcium iminodisuccinate:

[0134] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then a 25% ammonia solution is added, the amount of ammonia added is 0.8% of the mass fraction of water, and then 1.1 mole of calcium hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 88°C, heated under reflux at normal pressure for 32 hours, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120°C. The mass percentage of iminodisuccinate in the solid is 75%, and the mass percentage of calcium is 20%.

[0135] Zinc iminodisuccinate:

[0136] 0.8 mole of maleic anhydride and 0.8 mole of L-aspartic acid are added to 20 mole of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.8% of the mass fraction of water, and then 1.2 mole of zinc hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, the mixture is heated to 88° C., and heated under reflux for 30 hours at normal pressure. After the reaction is completed, the mixture is cooled to room temperature, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain solid zinc iminodisuccinate. The mass percentage of iminodisuccinate in the solid is 76%, and the mass percentage of zinc is 20%.

[0137] Manganese iminodisuccinate:

[0138] 0.9 molar parts of maleic anhydride and 0.9 molar parts of L-aspartic acid are added to 20 molar parts of water, and then ammonia solution with a mass concentration of 25% is added, and the amount of ammonia added is 0.8% of the mass fraction of water, and then 1.2 molar parts of manganese hydroxide is slowly added and stirred, and after the raw materials are completely dissolved, they are heated to 80° C., heated under reflux at normal pressure for 28 hours, and cooled to room temperature after the reaction is completed, the insoluble matter is filtered, and the solution is dried at 120° C. to obtain iminodisuccinate manganese solid. The mass percentage of iminodisuccinate in the solid is 76%, and the mass percentage of manganese is 20%.

[0139] The remaining parameters and preparation methods are the same as those in Example 1.

[0140] In this example, only the concentration of the prepared iminodisuccinate is slightly different from that in Example 1. It can be predicted that the effect of the fermentation medium prepared by using the same on the high-density fermentation culture of Escherichia coli is also similar to that in Example 1.

[0141] Experimental Results

[0142] Tables 1 to 7 are fermentation data tables of Comparative Examples 1-3 and Examples 1-4.

[0143] Figure 1 OD of each group of Comparative Examples 1-3 and Examples 1-4 600 Comparison chart with fermentation time.

[0144] Figure 2 The fermentation medium prepared in Example 1 and Comparative Example 1. Figure a is the fermentation medium prepared in Example 1, and Figure b is the fermentation medium prepared in Comparative Example 1.

[0145] Figure 3 This is a shake flask culture diagram of Example 1 of the present invention.

[0146] Figure 4 This is a fermentation culture diagram of Example 1 of the present invention.

[0147] Figure 5 It is a comparison diagram of the fermentation broth of Example 1 and Comparative Example 1. Among them, Figure a is the fermentation broth of Example 1, and Figure b is the fermentation broth of Comparative Example 1.

[0148] Table 1 Comparative Example 1 Fermentation Data Table

[0149] Time / h Temperature / ℃ Dissolved oxygen / % Flow rate / (L / min) <![CDATA[OD 600 ]]> pH Speed ​​(r / min) Feed / (ml / min) 0 37.0 90 3 6.91 200 0 1 37.0 82 3 7.05 200 0 2 36.7 69 3 7.08 200 0 3 36.6 61 3→5 7.09 200 0 4 36.7 43 5→7 7.03 200 0 5 37.1 40 7 7.00 260 0 6 37.0 59 7→10 12.2 6.95 320 0.28 7 37.0 42 10 16.6 6.92 320 0.28 8 37.0 40 10 22.2 6.92 340 0.32 9 37.0 40 10→7 24.8 6.91 340 0.44 10 37.1 41 7 35.6 6.93 380 0.44 11 37.0 40 7 47.6 6.98 440 0.53 12 37.0 41 7 56.2 6.97 480 0.53 13 37.1 40 7→9 64.6 7.02 400 0.53 14 37.0 40 9 70.0 7.04 580 0.53 15 37.0 40 9 78.0 6.83 681 0.53 16 37.0 41 9 88.0 6.60 761 0.53 17 37.0 42 9 89.6 6.90 922 0.53 18 37.0 40 9→11 95.2 6.91 932 0.53 19 37.1 45 11 96 6.91 953 0.53 20 37.0 42 11 103 6.90 974 0.66 21 37.1 42 11 106.8 6.93 974 0.66 22 37.0 41 11 111.2 6.95 974 0.66 23 37.0 43 11 117.6 6.91 974 0.66 24 37.0 40 11 125.2 6.92 974 0.66 25 37.1 40 11→12 122.2 6.91 974 0.66 26 37.1 40 12 126.2 6.91 974 0.66 27 37.0 45 12 132.8 6.97 974 0.66 28 37.0 48 12 132.2 6.91 974 0.66 29 37.1 42 12 132.2 6.92 974 0.66

[0150] Table 2 Fermentation data table of Example 1

[0151]

[0152]

[0153] Table 3 Comparative Example 2 Fermentation Data Table

[0154]

[0155]

[0156] Table 4 Fermentation data table of Example 2

[0157] Time / h Temperature / ℃ Dissolved oxygen / % Flow rate / (L / min) <![CDATA[OD 600 ]]> pH Speed ​​(r / min) Feed / (ml / min) 0 37.0 86 3 6.97 200 1 37.0 41 3→7 6.99 220 2 36.8 45 7 6.93 340 3 37.0 40 7 6.97 540 4 37.0 37 7 6.90 661 5 37.0 59 7 11.2 6.93 822 0.32 6 37.0 30 7→11 21.5 7.02 925 0.44 7 37.1 27 11 30.9 6.90 925 0.53 8 37.0 25 11 32.0 6.91 975 0.59 9 37.0 10 11 45.6 6.90 975 0.75 10 37.0 9 11 46.5 6.91 973 0.75 11 37.0 7 11→12 60.9 6.89 973 0.75 12 37.0 7 12 64.6 6.93 973 0.66 13 37.1 7 12 70.8 6.93 973 0.66 14 37.0 7 12 79.2 6.90 973 0.53 15 37.0 7 12 91.5 6.93 973 0.53 16 37.0 7 12 100.7 6.90 973 0.53 17 37.1 7 12 130.1 6.90 973 0.53 18 37.1 7 12 111.0 6.97 973 0.59 19 37.1 7 12 113.8 6.90 973 0.59 20 37.0 7 12 126.0 6.94 973 0.59 21 37.1 13 12 125.2 6.92 973 0.59 22 37.0 27 12 132.8 6.90 972 0.59 23 37.0 27 12 127.0 6.96 972 0.59 24 37.1 21 12 132.6 6.90 972 0.59 25 37.1 28 12 132.0 6.92 972 0.59

[0158] Table 5 Comparative Example 3 Fermentation Data Table

[0159]

[0160]

[0161] Table 6 Fermentation data table of Example 3

[0162]

[0163]

[0164] Table 7 Fermentation data table of Example 4

[0165] Time / h Temperature / ℃ Dissolved oxygen / % Flow rate / (L / min) <![CDATA[OD 600 ]]> pH Speed ​​(r / min) Feed / (ml / min) 0 37.0 81 3 6.99 200 1 37.0 49 3→7 7.05 221 2 37.0 42 7 7.04 325 3 36.9 41 7 6.95 515 4 37.1 41 7 6.95 640 5 37.0 41 7 6.91 750 6 37.0 62 7→9 12.0 7.11 455 0.32 7 37.0 49 9→12 18.8 7.00 875 0.59 8 37.1 44 12 25.6 6.95 735 0.66 9 37.0 44 12 39.2 6.89 955 0.66 10 37.0 40 12 44.7 6.91 950 0.75 11 37.1 41 12 56.8 6.90 955 0.75 12 37.1 40 12 62.2 6.91 970 0.87 13 37.0 42 12 70.1 6.90 970 0.87 14 37.0 42 12 75.5 6.92 970 0.30 15 37.0 41 12 75.5 7.10 910 0.53 16 37.0 40 12 81.2 7.13 970 0.53 17 37.0 42 12 84.0 6.91 970 0.56 18 37.1 42 12 88.4 6.90 970 0.59 19 37.0 40 12 92.6 6.90 970 0.59 20 37.0 41 12 99.5 6.93 970 0.59 21 37.0 41 12 104.8 6.91 970 0.59 22 37.0 42 12 112.8 6.90 970 0.59 23 37.0 41 12 115.6 6.91 972 0.59 24 37.1 40 12 120.6 6.92 970 0.59 25 37.1 42 12 126.4 6.92 970 0.59 26 37.1 44 12 128.5 6.91 970 0.59 27 37.0 41 12 135.5 6.92 970 0.59 28 37.1 40 12 135.5 6.92 970 0.59 29 37.0 42 12 136.1 6.91 970 0.59 30 37.0 41 12 136.0 6.92 970 0.59

[0166] From Tables 1 to 7 and Figure 1 It can be seen that:

[0167] Comparative Example 1 used inorganic salts, dissolved oxygen rebounded after 6 hours, and feeding began, reaching the highest biomass OD in 27 hours 600 =132.8.

[0168] Example 1: Using iminodisuccinate, dissolved oxygen rebounded after 6 hours, feeding began, and the maximum biomass OD was reached after 26 hours. 600 ==141.9.

[0169] Comparative Example 2 used inorganic salts, dissolved oxygen rebounded after 7 hours, and feeding began, reaching the highest biomass OD in 23 hours 600 =122.4.

[0170] Example 2: Using iminodisuccinate, dissolved oxygen rebounded after 5 hours, feeding began, and the maximum biomass OD was reached after 22 hours. 600 ==132.8.

[0171] Comparative Example 3 used inorganic salts, dissolved oxygen rebounded after 4 hours, and feeding began. The highest biomass OD was reached in 25 hours. 600 =96.0.

[0172] Example 3: Using iminodisuccinate, dissolved oxygen rebounded after 6 hours, feeding began, and the maximum biomass OD was reached after 27 hours. 600 ==127.0.

[0173] Example 4: Using iminodisuccinate, dissolved oxygen rebounded after 6 hours, feeding began, and the highest biomass OD was reached after 29 hours. 600 ==136.1.

[0174] By comparing the fermentation of Comparative Example 1 and Example 1 (Example 1 is the optimal trace element concentration), the fermentation data shows that under the same trace element usage conditions, the use of inorganic salts reaches the highest biomass OD in 27 hours.600 = 132, and the highest biomass OD was reached in 26 hours using iminodisuccinate 600 = about 141. This shows that the fermentation using iminodisuccinate is faster than that using inorganic salts, and the final biomass is higher.

[0175] Comparative Example 2 and Example 2 used lower trace element concentrations for fermentation, and the conclusions obtained were consistent with those of Comparative Example 1 and Example 1.

[0176] Comparative Example 3 and Example 3 used higher trace element concentrations for fermentation. Comparative Example 3 reached the highest biomass OD at 25 hours. 600 =96.0, Example 3 reached the highest biomass OD at 27 hours 600 = = 127.0. Although the time to reach the highest biomass in Comparative Example 3 was shorter, the reason was that the higher element concentration greatly reduced the fermentation level of Escherichia coli, resulting in a shortened fermentation cycle and a lower fermentation yield, while iminodisuccinate greatly reduced this effect.

[0177] By comparing Comparative Example 1 and Comparative Example 2 with Example 1 and Example 2, it can be found that within a certain concentration range, the reduction of trace element dosage will shorten the logarithmic growth period of Escherichia coli and reduce the final biomass of fermentation. The use of iminodisuccinate can improve the utilization rate of trace elements, extend the logarithmic growth period, and reduce the impact of insufficient trace elements on the final fermentation results.

[0178] By comparing Comparative Example 1 and Comparative Example 3 with Example 1 and Example 3, it can be found that excessive trace elements are not conducive to the fermentation results. Excessive trace elements will significantly reduce the final biomass of the fermentation. Although the fermentation results will also be reduced when iminodisuccinate is used instead of inorganic salts, the degree of reduction is much lower than that of using organic salts.

[0179] By comparing Comparative Example 1 and Example 2, it can be found that the concentration of trace elements in Example 2 is lower, but the final fermentation results of the two are basically the same, and the fermentation speed of Example 2 is faster, indicating that iminodisuccinate is more conducive to Escherichia coli fermentation than inorganic salts.

[0180] By comparing Comparative Example 1 with Example 1, Comparative Example 2 with Example 2, and Comparative Example 3 with Example 3, it can be found that when the concentration of trace elements is the same, the final biomass of the fermentation of iminodisuccinate is increased compared with normal inorganic salts.

[0181] By comparing Example 1 and Example 4, it can be found that within the optimum concentration range, when using organic trace elements, appropriately reducing the trace element concentration has little effect on the final fermentation result. When the trace element concentration is reduced by 10%, the maximum OD 600It decreased from 141.9 to 136.1. This indicates that the use of iminodisuccinate can regulate the use of trace elements by Escherichia coli within a certain range, making it more useful for fermentation production.

[0182] The above examples show that in the high-density fermentation process of Escherichia coli, iminodisuccinate is more easily absorbed and utilized by microorganisms than normal inorganic salts, thereby increasing the fermentation biomass and being more conducive to fermentation production.

[0183] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of iminodisuccinate in high-density fermentation of microorganisms; the iminodisuccinate comprises at least one of copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate and manganese iminodisuccinate.

2. The use according to claim 1, characterized in that: The preparation method of the iminodisuccinate is as follows: maleic anhydride and L-aspartic acid are added to water and an ammonia solution, and then a metal hydroxide is added and stirred, and after being completely dissolved, the metal hydroxide is heated to 75-95° C., and refluxed under normal pressure for reaction, and after the reaction is completed, the solution is dried to obtain an iminodisuccinate solid; the metal hydroxide includes copper hydroxide, ferrous hydroxide, calcium hydroxide, zinc hydroxide and manganese hydroxide.

3. The use according to claim 1 or 2, characterized in that: The microorganism is Escherichia coli.

4. The use according to claim 1 or 2, characterized in that: In the high-density fermentation of microorganisms, the iminodisuccinate is used as a trace element in the microorganism fermentation medium.

5. The use according to claim 4, characterized in that: In the microbial fermentation medium, the concentration of the copper element is 0.0130-0.0280 g / L; and / or The concentration of the ferrous element is 0.0210-0.0440 g / L; and / or The concentration of the calcium element is 0.0060-0.0170 g / L; and / or The concentration of the zinc element is 0.0150-0.0260 g / L; and / or The concentration of the manganese element is 0.0012-0.0028 g / L.

6. The use according to claim 5, characterized in that: In the microbial fermentation medium, the concentration of the copper element is 0.0153-0.0255 g / L; the concentration of the ferrous element is 0.0241-0.0402 g / L; the concentration of the calcium element is 0.0072-0.0144 g / L; the concentration of the zinc element is 0.0182-0.0227 g / L; and the concentration of the manganese element is 0.0015-0.0025 g / L.

7. The use according to claim 6, characterized in that: In the microbial fermentation medium, the concentration of the copper element is 0.0204 g / L; the concentration of the ferrous element is 0.0321 g / L; the concentration of the calcium element is 0.0108 g / L; the concentration of the zinc element is 0.0205 g / L; and the concentration of the manganese element is 0.0020 g / L.

8. A fermentation medium for high-density fermentation of microorganisms, characterized in that: The fermentation medium comprises a trace element mother solution, wherein the formula of the trace element mother solution is: copper iminodisuccinate, ferrous iminodisuccinate, calcium iminodisuccinate, zinc iminodisuccinate, and manganese iminodisuccinate; In the microbial fermentation medium, the concentration of the copper element is 0.0153-0.0255 g / L; the concentration of the ferrous element is 0.0241-0.0402 g / L; the concentration of the calcium element is 0.0072-0.0144 g / L; the concentration of the zinc element is 0.0182-0.0227 g / L; and the concentration of the manganese element is 0.0015-0.0025 g / L.

9. A fermentation medium for high-density fermentation of microorganisms according to claim 8, characterized in that: In the microbial fermentation medium, the concentration of the copper element is 0.0204 g / L; the concentration of the ferrous element is 0.0321 g / L; the concentration of the calcium element is 0.0108 g / L; the concentration of the zinc element is 0.0205 g / L; and the concentration of the manganese element is 0.0020 g / L.

10. A method for high-density fermentation and cultivation of microorganisms, characterized in that: The following steps are involved: (1) activating the microbial strain and performing primary strain culture and secondary seed culture; preferably, the microorganism is Escherichia coli; (2) Fermentation culture: The secondary seeds are inoculated into a fermentation tank at an inoculation rate of 2%-8%, and fermentation is carried out using the fermentation medium described in claim 8 or 9, the rotation speed is coupled with the ventilation volume and dissolved oxygen, the dissolved oxygen is controlled to be ≥ 40% of the initial oxygen content, and the pH is controlled to be 6.8-7.2; after the dissolved oxygen rebounds, feeding is started, and fermentation is carried out until the fermentation liquid OD 600 No more growth, fermentation ends; Preferably, the feed medium during feeding is: 300-600 g / L glucose, 8-16 g / L peptone, 8-16 g / L yeast powder, and 3-10 g / L magnesium sulfate heptahydrate.

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