Denitrification hyphomicrobium JCSS230201 and application thereof in production of pyrroloquinoline quinone

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a denitrifying hyphomicrobium JCSS230201 strain and application of the denitrifying hyphomicrobium JCSS230201 strain in production of pyrroloquinoline quinone. The denitrifying hyphomicrobe JCSS230201 obtained through screening is a natural high-yield pyrroloquinoline quinone strain, pyrroloquinoline quinone can be efficiently produced in a culture medium only containing methanol and inorganic salt without a complex regulation and control process, experimental results show that the yield of pyrroloquinoline quinone reaches 1056.9 mg / L after fermentation is carried out for 182 h under the condition of the invention, and the yield of pyrroloquinoline quinone reaches 1056.9 mg / L. The natural strain is combined with a low-cost process, and a technical approach which is more economical, stable and easy for industrial preparation is provided for PQQ.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a strain of Hyphomicrobium denitrificans JCSS230201 and its application in the production of pyrroloquinoline quinone. Background Art

[0002] Pyrroloquinoline quinone (PQQ) is a novel redox coenzyme. PQQ was initially discovered as a coenzyme of bacterial methanol dehydrogenase, and subsequently detected in various microorganisms, plants, and even some animal tissues. However, the PQQ content in these organisms is usually very low.

[0003] Numerous studies have shown that PQQ exhibits a wide range of physiological activities in promoting cell proliferation, organism growth, antioxidant activity, improving mitochondrial function, anti-aging, and enhancing immunity, making it have great application potential in the fields of health products, cosmetics, medicine, and agriculture.

[0004] Currently, the industrial production of PQQ mainly relies on microbial fermentation, which has the advantages of being more environmentally friendly, low-cost, and natural compared to chemical synthesis. Microorganisms capable of producing PQQ mainly include methylotrophic bacteria such as Hyphomicrobium denitrificans and Methylobacterium extorquens.

[0005] To meet the market demand for PQQ and further improve the production efficiency of PQQ, existing research mainly focuses on the following two aspects: First, obtaining high-yield strains through mutagenesis breeding or genetic engineering means. For example, Patent CN115449499A discloses Hyphomicrobium denitrificans MQ004-2 obtained by ARTP and NTG mutagenesis, Patent CN117946900A discloses Hyphomicrobium denitrificans HRY-0816 obtained by ARTP mutagenesis, and Patent CN117778273A discloses Methylobacterium extorquens B70 obtained by ultraviolet mutagenesis. These mutagenized strains combined with corresponding fermentation processes have indeed improved the PQQ yield to a certain extent, and some reported yields have exceeded 2 g / L. However, mutagenesis breeding often relies on specific mutagenesis equipment or reagents, and may also be accompanied by problems such as uncertain genetic stability of the strains and low screening efficiency.

[0006] Second, improving the fermentation level by optimizing the fermentation process control and medium components. For example, Patent CN116536376A proposes a method for dynamically regulating the methanol concentration according to the morphology of Hyphomicrobium. Although it can improve the PQQ productivity, it is relatively complex to monitor the cell morphology in large-scale production, and the operability faces challenges.

[0007] In addition, some technical solutions increase the production by adding specific promoters such as glutamic acid, serine (CN117946900A), or amino acids such as proline and arginine (CN117778273A) to the culture medium, or adopting more complex strategies such as using lignocellulose hydrolysate as a substrate and adding various cofactors such as enzyme inhibitors (phenoxyquinoline) (CN118308441A). Although the production can be increased, it undoubtedly increases the complexity of the process. Patent CN118006517A explores the use of CO2 autotrophic bacteria for production, but the reported production is relatively low at present, and there is still a long way to industrial application.

[0008] Although the existing technologies have made certain progress in increasing the production of PQQ, they are often accompanied by problems such as risks of strain instability, increased process complexity, and elevated production costs.

[0009] There is still an urgent need in this field for a new method for producing PQQ that can balance production, cost, operational simplicity, and strain stability. In particular, exploring and utilizing microbial resources with natural high-production potential, stable genetic traits, and capable of efficiently synthesizing PQQ under conventional fermentation conditions, along with their supporting simplified and low-cost fermentation processes, will be an important breakthrough direction in the field of PQQ biomanufacturing. Summary of the Invention

[0010] To solve the above technical problems, the present invention provides a strain of Hyphomicrobium denitrificans JCSS230201 and its application in the production of pyrroloquinoline quinone.

[0011] The Hyphomicrobium denitrificans JCSS230201 provided by the present invention is a natural strain obtained through screening. Without complex regulation processes, this strain can efficiently ferment and produce pyrroloquinoline quinone in a culture medium containing only methanol and inorganic salts. The combination of this natural strain and a low-cost process provides a more economical, stable, and easily industrializable technical route for PQQ.

[0012] The first object of the present invention is to provide a strain of Hyphomicrobium denitrificans JCSS230201, and the said Hyphomicrobium denitrificans ( Hyphomicrobium denitrificans ) JCSS230201 was deposited on February 8, 2025, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit address being: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number being CGMCC No. 33506, and the taxonomic name being: Hyphomicrobium denitrificans Hyphomicrobium denitrificans .

[0013] The physical and chemical properties of the denitrifying Hyphomicrobium JCSS230201 are as follows: Gram-negative bacteria, with small colonies, round in shape, smooth surface, and the colony color is milky white or light yellow. Under an optical microscope, the cell morphology is spindle-shaped.

[0014] The second object of the present invention is to provide the application of the denitrifying Hyphomicrobium JCSS230201 in the production of pyrroloquinoline quinone.

[0015] The third object of the present invention is to provide a method for producing pyrroloquinoline quinone, and the method specifically includes the following steps: (1) Seed culture: Inoculate the denitrifying Hyphomicrobium JCSS230201 into a seed medium for primary and secondary seed cultures to obtain a seed solution of the denitrifying Hyphomicrobium JCSS230201; Among them, the conditions for primary seed culture are: stirring speed 200 - 240 rpm, temperature 28 - 30 °C, fermentation duration 20 - 24 h; The conditions for secondary seed culture are: seed tank volume 3.5 L / 5 L, inoculation amount 2% - 5%, culture temperature 28 - 30 °C, ventilation 3 - 4 L / h, rotation speed constant at 180 - 220 rpm, fermentation duration 40 - 50 h; (2) Fermentation culture: Inoculate the seed solution of the denitrifying Hyphomicrobium JCSS230201 cultured in (1) into a fermentation medium for fermentation culture to obtain the pyrroloquinoline quinone; The conditions for fermentation culture are: inoculation amount 2% - 8%, stirring speed 100 - 200 rpm, temperature 28 - 30 °C, ventilation 400 - 600 L / h, dissolved oxygen 20% - 60%, methanol feeding rate 30 - 60 g / h, controlling the methanol concentration in the fermentation broth to be less than 0.5 g / L, and fermentation time 168 - 240 h.

[0016] Preferably, the seed medium described in (1) contains the following components: anhydrous methanol 10 - 30 mL / L, ammonium sulfate 2 - 5 g / L, potassium dihydrogen phosphate 1 - 2 g / L, disodium hydrogen phosphate dodecahydrate 7 - 8 g / L, magnesium sulfate heptahydrate 1 - 2 g / L, 0.525% ferrous sulfate heptahydrate 1 - 3 mL / L, trace element A 0.7 mL / L, trace element B 0.7 mL / L, trace element C 0.7 - 0.8 mL / L, pH 6.0 - 7.2; Among them, the formulation of trace element A is: zinc sulfate heptahydrate 7 - 15 g / L, manganese sulfate monohydrate 1 - 3 g / L, copper sulfate pentahydrate 0.3 - 0.75 g / L; the formulation of trace element B is: sodium chloride 1.2 - 1.6 g / L, cobalt chloride hexahydrate 10 - 20 mg / L, calcium chloride 15 - 25 g / L; the formulation of trace element C is: ammonium molybdate tetrahydrate 10 - 25 mg / L, potassium iodide 15 - 25 mg / L, boric acid 28 - 32 mg / L.

[0017] Preferably, the fermentation medium described in (2) comprises the following components: Fermentation medium: anhydrous methanol 10 - 30 mL / L, ammonium sulfate 2 - 5 g / L, potassium dihydrogen phosphate 1 - 2 g / L, disodium hydrogen phosphate dodecahydrate 6 - 8 g / L, magnesium sulfate heptahydrate 1 - 3 g / L, 0.525% ferrous sulfate heptahydrate 1 - 3 mL / L, trace element A 0.7 mL / L, trace element B 0.7 mL / L, trace element C 0.7 - 0.8 mL / L, pH 6.0 - 7.2; Among them, the formulation of trace element A is: zinc sulfate heptahydrate 7 - 15 g / L, manganese sulfate monohydrate 1 - 3 g / L, copper sulfate pentahydrate 0.3 - 0.75 g / L; the formulation of trace element B is: sodium chloride 1.3 - 1.7 g / L, cobalt chloride hexahydrate 10 - 20 mg / L, calcium chloride 15 - 25 g / L; the formulation of trace element C is: ammonium molybdate tetrahydrate 10 - 25 mg / L, potassium iodide 15 - 25 mg / L, boric acid 25 - 35 mg / L.

[0018] The beneficial effects of the present invention are as follows: The denitrifying Hyphomicrobium JCSS230201 provided by the present invention can efficiently ferment and synthesize pyrroloquinoline quinone in a medium containing only methanol and inorganic salts. Moreover, through the optimization of conditions, the optimal yield of pyrroloquinoline quinone can reach 1056.9 mg / L.

[0019] Compared with the prior art, by using the denitrifying Hyphomicrobium JCSS230201 provided by the invention, pyrroloquinoline quinone can be efficiently produced in a medium containing only methanol and inorganic salts without complex fermentation media and regulation processes, and has broad industrial application prospects. Description of the Drawings

[0020] Figure 1 It is the slant culture and microscope photograph of the denitrifying Hyphomicrobium JCSS230201 provided by the present invention, wherein (A) is the slant culture of the denitrifying Hyphomicrobium JCSS230201, and (B) is the microscope photograph of the denitrifying Hyphomicrobium JCSS230201; Figure 2 It is the HPLC chromatogram of the pyrroloquinoline quinone standard; Figure 3 This is the HPLC chromatogram of pyrroloquinoline quinone in the fermentation broth of Hyphomicrobium denitrificans JCSS230201 obtained by the method of the present invention in Example 3. Detailed implementation manners

[0021] In order to enable those skilled in the art to better understand the present invention, the present invention will be further elaborated below in conjunction with specific implementation manners.

[0022] Example 1 Screening of strains Soil samples were collected from the mountain of Yuanshan Building in Boshan, Shandong Province. After making a suspension, they were spread on an enrichment medium (anhydrous methanol mass fraction is 1%, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 1.4 g / L, disodium hydrogen phosphate dodecahydrate 7.56 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate with a mass fraction of 0.525% 1 mL / L, agar 20 g / L). Then, the bacterial liquid in the enrichment medium was diluted with sterile water and spread on a screening medium (anhydrous methanol 2%, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 1.4 g / L, disodium hydrogen phosphate dodecahydrate 7.56 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 0.525% 1 mL / L, agar 20 g / L), and cultured at 30 °C for 3 - 5 days.

[0023] The obtained culture was transferred to a new solid plate, and streak plating was carried out until single colonies appeared. After rescreening by detecting the yield of β-hydroxy-β-methylbutyric acid (HMB) in a triangular flask fermentation, a pure culture, a strain numbered JCSS230201, was finally obtained.

[0024] The JCSS230201 strain was identified by 16S rDNA. By constructing an evolutionary tree with partial sequences obtained from NCBI blast, it was determined that JCSS230201 is Hyphomicrobium denitrificans Hyphomicrobium denitrificans .

[0025] The obtained Hyphomicrobium denitrificans JCSS230201 was deposited on February 8, 2025, at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No. 33506, and the taxonomic name: Hyphomicrobium denitrificans Hyphomicrobium denitrificans .

[0026] Appendix Figure 1 Shows the slant view and microscope photograph of Hyphomicrobium denitrificans JCSS230201 provided by the present invention, where (A) is the slant view of Hyphomicrobium denitrificans JCSS230201, and (B) is the microscope photograph of Hyphomicrobium denitrificans JCSS230201.

[0027] As can be seen in the figure, the denitrifying Hyphomicrobium JCSS230201 is a Gram-negative bacterium. Its colonies are small, round, with a smooth surface, and the colony color is milky white or light yellow. Under an optical microscope, the cell morphology is spindle-shaped.

[0028] Example 2: Fermentation of Pyrroloquinoline Quinone by Denitrifying Hyphomicrobium JCSS230201 The denitrifying Hyphomicrobium JCSS230201 obtained by screening in Example 1 was subjected to multi-batch fermentation culture to produce pyrroloquinoline quinone. The specific operations are as follows: (1) Seed culture: The denitrifying Hyphomicrobium JCSS230201 was inoculated into a seed medium for first-stage and second-stage seed cultures to obtain a seed solution of denitrifying Hyphomicrobium JCSS230201. Among them, the conditions for the first-stage seed culture are: stirring speed of 200 rpm, temperature of 28 °C, and fermentation for 24 h. The conditions for the second-stage seed culture are: a 3.5L / 5L seed tank (i.e., the volume of the seed tank is 5L, and the liquid loading is 3.5L), an inoculation amount of 100 mL, a culture temperature of 28 °C, a ventilation rate of 3.5 L / h, a constant rotation speed of 200 rpm, and a fermentation time of 42 h. The composition of the seed medium is: anhydrous methanol 12 mL / L, ammonium sulfate 2 g / L, potassium dihydrogen phosphate 1.2 g / L, disodium hydrogen phosphate dodecahydrate 7.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, 0.525% ferrous sulfate heptahydrate 1 mL / L, trace element A 0.7 mL / L, trace element B 0.7 mL / L, trace element C 0.8 mL / L, pH 6.9 - 7.1. Specifically, the formula of trace element A is: zinc sulfate heptahydrate 10 g / L, manganese sulfate monohydrate 1.5 g / L, copper sulfate pentahydrate 0.75 g / L; the formula of trace element B is: sodium chloride 1.5 g / L, cobalt chloride hexahydrate 20 mg / L, calcium chloride 18 g / L; the formula of trace element C is: ammonium molybdate tetrahydrate 18 mg / L, potassium iodide 25 mg / L, boric acid 30 mg / L. (2) Fermentation culture: The seed solution of denitrifying Hyphomicrobium JCSS230201 cultured in (1) was inoculated into a fermentation medium for fermentation culture to obtain the pyrroloquinoline quinone. The conditions for fermentation culture are: an inoculation amount of 4%, a stirring speed of 150 rpm, a temperature of 28 °C, a ventilation rate of 450 L / h, a dissolved oxygen of 30%, a methanol feeding rate of 40 g / h, and controlling the methanol concentration in the fermentation broth to be less than 0.5 g / L.

[0029] Fermentation medium: anhydrous methanol 16 mL / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 1.4 g / L, disodium hydrogen phosphate dodecahydrate 7.56 g / L, magnesium sulfate heptahydrate 1 g / L, ferrous sulfate heptahydrate 0.525% 1 mL / L, trace element A 0.7 mL / L, trace element B 0.7 mL / L, trace element C 0.7 mL / L, pH 7.0 - 7.2; Specifically, the formula of trace element A is: zinc sulfate heptahydrate 7.0 g / L, manganese sulfate monohydrate 1.0 g / L, copper sulfate pentahydrate 0.5 g / L; the formula of trace element B is: sodium chloride 1.5 g / L, cobalt chloride hexahydrate 20 mg / L, calcium chloride 15 g / L; the formula of trace element C is: ammonium molybdate tetrahydrate 22 mg / L, potassium iodide 22 mg / L, boric acid 30 mg / L.

[0030] The pyrroloquinoline quinone in the fermentation broth was detected by HPLC, and the specific method is as follows: Fermentation sample treatment: Dilute the fermentation broth by 10 times and centrifuge at 12000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm aqueous filter membrane.

[0031] HPLC detection method: Chromatographic column Inertsil ODS 4 (5um 4.6*250 mm), ultraviolet detector, detection wavelength 259 nm, column temperature 40 °C, flow rate 1.5 mL / min, injection volume 20 uL.

[0032] Mobile phase preparation: Weigh 5.75 mL (6.035 g) of acetic acid and dissolve it in 1 L of water to obtain 0.1 M acetic acid solution; weigh 7.708 g of ammonium acetate and dissolve it in 1 L of water to obtain 0.1 M ammonium acetate solution; prepare the mobile phase according to the volume ratio of 0.1 M acetic acid∶0.1 M ammonium acetate = 30∶70 (pH = 5.1).

[0033] The HPLC chromatogram of the pyrroloquinoline quinone standard is shown in the appendix Figure 2 as shown.

[0034] After 196 h of fermentation, the methanol consumption was 6500 mL, and the yield of pyrroloquinoline quinone in the fermentation broth was 785.1 mg / L detected by HPLC.

[0035] Example 3 Fermentation of Hyphomicrobium denitrificans JCSS230201 The Hyphomicrobium denitrificans JCSS230201 obtained by screening in Example 1 was subjected to multi-batch fermentation culture to produce pyrroloquinoline quinone, and the specific operation is as follows: (1)Seed culture: Inoculate Hyphomicrobium denitrificans JCSS230201 into the seed medium for primary and secondary seed cultures to obtain a seed solution of Hyphomicrobium denitrificans JCSS230201; Primary seed culture conditions: Stirring speed is 240 rpm, temperature is 30 °C, and fermentation lasts for 24 h; Secondary seed culture conditions: The volume of the seed tank is 3.5 L / 5 L, the inoculum size is 100 mL, the culture temperature is 30 °C, the ventilation is 3.5 L / L, the rotation speed is constantly 200 rpm, and the fermentation time is 43 h.

[0036] Seed medium: 16 mL / L of anhydrous methanol, 3 g / L of ammonium sulfate, 1.4 g / L of potassium dihydrogen phosphate, 7.56 g / L of disodium hydrogen phosphate dodecahydrate, 1 g / L of magnesium sulfate heptahydrate, 1 mL / L of 0.525% ferrous sulfate heptahydrate, 0.7 mL / L of trace element A, 0.7 mL / L of trace element B, 0.7 mL / L of trace element C, pH 6.8 - 7.0; Specifically, the formula of trace element A is: 15 g / L of zinc sulfate heptahydrate, 3 g / L of manganese sulfate monohydrate, 0.75 g / L of copper sulfate pentahydrate; the formula of trace element B is: 1.5 g / L of sodium chloride, 20 mg / L of cobalt chloride hexahydrate, 25 g / L of calcium chloride; the formula of trace element C is: 25 mg / L of ammonium molybdate tetrahydrate, 25 mg / L of potassium iodide, 30 mg / L of boric acid; (2)Fermentation culture: Inoculate the seed solution of Hyphomicrobium denitrificans JCSS230201 cultured in (1) into the fermentation medium for fermentation culture to obtain the pyrroloquinoline quinone; Fermentation conditions: Inoculum size is 4%, stirring speed is 200 rpm, temperature is 30 °C, ventilation is 600 L / h, dissolved oxygen is 40%, the methanol feeding rate is 60 g / h, and the methanol concentration in the fermentation broth is controlled to be less than 0.5 g / L.

[0037] Fermentation medium: 16 mL / L of anhydrous methanol, 3 g / L of ammonium sulfate, 1.4 g / L of potassium dihydrogen phosphate, 7.56 g / L of disodium hydrogen phosphate dodecahydrate, 1 g / L of magnesium sulfate heptahydrate, 1 mL / L of 0.525% ferrous sulfate heptahydrate, 0.7 mL / L of trace element A, 0.7 mL / L of trace element B, 0.7 mL / L of trace element C, pH 6.8 - 7.0; Specifically, the formulation of trace element A is: zinc sulfate heptahydrate 10 g / L, manganese sulfate monohydrate 2.0 g / L, copper sulfate pentahydrate 0.5 g / L; the formulation of trace element B is: sodium chloride 1.5 g / L, cobalt chloride hexahydrate 15 mg / L, calcium chloride 20 g / L; the formulation of trace element C is: ammonium molybdate tetrahydrate 20 mg / L, potassium iodide 20 mg / L, boric acid 30 mg / L.

[0038] The HPLC chromatogram of pyrroloquinoline quinone in the fermentation broth of the denitrifying Hyphomicrobium JCSS230201 obtained in this example is shown in the appendix Figure 3 as follows.

[0039] After 182 h of fermentation with a methanol dosage of 7500 mL, the yield of pyrroloquinoline quinone in the fermentation broth was 1056.9 mg / L.

[0040] The above embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of this patent, several deformations, combinations, and improvements can be made to the above embodiments, and these all belong to the protection scope of the present invention.

Claims

1. A denitrifying mycelial microorganism JCSS230201, characterized in that: The denitrifying filamentous microorganism ( Hyphomicrobium denitrificans )JCSS230201, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on February 8, 2025, with the deposit number: CGMCC No.33506, and the classification name: Denitrifying Mycelial Microbe Hyphomicrobium denitrificans .

2. Use of the denitrifying hyphae microbacterium JCSS230201 according to claim 1 in the production of pyrroloquinoline quinone.

3. A method for producing pyrroloquinoline quinone, characterized in that: The steps include: (1) Seed culture: inoculate the denitrifying mycelial microorganism JCSS230201 into a seed culture medium for primary and secondary seed culture to obtain a seed solution of the denitrifying mycelial microorganism JCSS230201; Among them, the conditions for primary seed culture are: stirring speed 200-240 rpm, culture temperature 28-30℃, fermentation time 20-24 h; The conditions for secondary seed culture are: seed tank volume 3.5 L / 5 L, inoculation amount 2%-5%, culture temperature 28-30℃, ventilation 3-4 L / h, constant speed 180-220 rpm, fermentation time 40-50 h; (2) Fermentation culture: inoculating the seed liquid of the denitrifying hyphae microbe JCSS230201 cultured in (1) into a fermentation medium for fermentation culture to obtain the pyrroloquinoline quinone; The fermentation culture conditions are: inoculation size 2%-8%, stirring speed 100-200 rpm, culture temperature 28-30°C, ventilation 400-600 L / h, dissolved oxygen 20%-60%, methanol feed rate 30-60 g / h, methanol concentration in the fermentation broth controlled to be less than 0.5 g / L, and fermentation time 168-240 h.

4. The method according to claim 3, characterized in that The seed culture medium described in (1) comprises the following components: anhydrous methanol 10-30 mL / L, ammonium sulfate 2-5 g / L, potassium dihydrogen phosphate 1-2 g / L, disodium hydrogen phosphate dodecahydrate 7-8 g / L, magnesium sulfate heptahydrate 1-2 g / L, 0.525% ferrous sulfate heptahydrate 1-3 mL / L, trace element A 0.7 mL / L, trace element B 0.7 mL / L, trace element C 0.7-0.8 mL / L, pH 6.0-7.2; Among them, the formula of trace element A is: zinc sulfate heptahydrate 7-15 g / L, manganese sulfate monohydrate 1-3 g / L, copper sulfate pentahydrate 0.3-0.75 g / L; the formula of trace element B is: sodium chloride 1.2-1.6 g / L, cobalt chloride hexahydrate 10-20 mg / L, calcium chloride 15-25 g / L; the formula of trace element C is: ammonium molybdate tetrahydrate 10-25 mg / L, potassium iodide 15-25 mg / L, boric acid 28-32 mg / L.

5. The method according to claim 3, characterized in that The fermentation medium described in (2) comprises the following components: Fermentation medium: anhydrous methanol 10-30 mL / L, ammonium sulfate 2-5 g / L, potassium dihydrogen phosphate 1-2 g / L, disodium hydrogen phosphate dodecahydrate 6-8 g / L, magnesium sulfate heptahydrate 1-3 g / L, 0.525% ferrous sulfate heptahydrate 1-3 mL / L, trace element A 0.7 mL / L, trace element B 0.7 mL / L, trace element C 0.7-0.8 mL / L, pH 6.0-7.2; Among them, the formula of trace element A is: zinc sulfate heptahydrate 7-15 g / L, manganese sulfate monohydrate 1-3 g / L, copper sulfate pentahydrate 0.3-0.75 g / L; the formula of trace element B is: sodium chloride 1.3-1.7 g / L, cobalt chloride hexahydrate 10-20 mg / L, calcium chloride 15-25 g / L; the formula of trace element C is: ammonium molybdate tetrahydrate 10-25 mg / L, potassium iodide 15-25 mg / L, boric acid 25-35 mg / L.

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