Method for producing bioethylene by using carbon dioxide

Abstract

The invention discloses a method for producing bioethylene by using carbon dioxide, and relates to the technical field of genetic engineering. A recombinant expression bacterium is obtained by transferring an ethylene forming enzyme coding gene efe with a nucleotide sequence as shown in SEQ ID NO.1 into a recombinant bacterium, and the obtained recombinant expression bacterium can express an ethylene synthase gene and stably inherit the ethylene synthase gene, so that a large batch of CO2 is absorbed, fixed and converted into bioethylene. The recombinant expression strain constructed by the invention can be used for producing bioethylene, a new carbon sequestration pathway is provided, the concentration of greenhouse gases can be effectively reduced, global warming can be relieved, the generated ethylene can also realize resource utilization of wastes, and the practical value is remarkable.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a method for producing bio-ethylene by utilizing carbon dioxide. Background Art

[0002] Today's society's overreliance on non-renewable energy sources such as oil and coal has led to serious environmental pollution and climate change, with CO2 emissions being the primary driver of global warming. To address this challenge, the development of novel CO2 capture and utilization technologies is imperative. Currently, mainstream CO2 treatment technologies include physical storage, chemical absorption, and biological carbon sequestration. Physical storage requires compressing CO2 and injecting it into the deep sea or rock formations, which is costly and difficult to scale. Chemical absorption, while more stable, carries the risk of reagent loss and secondary contamination. In contrast, biological carbon sequestration uses microorganisms to convert CO2 into organic matter, offering both environmental and sustainable advantages.

[0003] In this context, the construction of microbial cell factories using the chemoautotrophic hydrogen-producing bacterium Cupriavidus necator has become a highly promising solution. This strain naturally possesses the ability to utilize CO2 and H2 for growth. By genetically engineering the system to introduce exogenous metabolic pathways, such as ethylene synthase (e.g., the efe gene), it can be transformed into a biosynthetic platform for the efficient production of small-molecule compounds such as ethylene. Compared to traditional microbial production systems that rely on carbohydrate substrates, this technology offers three core advantages: first, it directly utilizes industrial waste gas (CO2) and green hydrogen as raw materials, significantly reducing production costs; second, it tolerates non-strictly sterile environments, simplifying the culture process; and third, by optimizing metabolic networks through synthetic biology, it enables efficient, targeted synthesis of products.

[0004] The breakthrough of this technology lies in its integration of CO2 resource utilization with green biomanufacturing, addressing greenhouse gas emissions while avoiding the reliance of traditional chemical production on fossil feedstocks. By developing gene editing toolkits (such as the CRISPR system) and optimizing expression vectors and regulatory elements, the production stability and economic efficiency of the strain can be further improved, providing an innovative paradigm for sustainable chemical production under the goal of carbon neutrality.

[0005] Through genetic engineering, C. necator , introducing exogenous ethylene synthase (EFE) into the metabolic pathway, enabling it to produce ethylene from CO2. Compared with traditional Escherichia coli, Zymomonas mobilis or cyanobacteria that rely on sugar substrates, the modified hydrogenotrophic strain has significant cost advantages: 1. Cheap raw materials: Directly using industrial waste gas (CO2) and hydrogen as carbon sources and energy, without the need for organic carbon sources (such as glucose) or lighting systems, significantly reducing substrate costs; 2. Simplified culture: Tolerant to open environments, no strict sterile conditions are required, reducing fermentation tank sterilization and operating costs; 3. High yield potential: Autotrophic growth can achieve high cell density (such as accumulation of PHB), combined with efe The efficient expression of genes and the ethylene production per unit volume are significantly better than other strains.

[0006] This patent seeks to develop a new technology for synthesizing ethylene through hydrogen bacteria, using CO2 and hydrogen as raw materials, replacing traditional high-cost sugars and hazardous chemicals to achieve safe and economical large-scale production. This will significantly reduce production costs, enhance industry competitiveness, and provide an innovative solution for green ethylene manufacturing. Summary of the Invention

[0007] The purpose of the present invention is to provide a method for producing bio-ethylene using CO2, which helps to neutralize and convert carbon dioxide and also provides a new way to produce ethylene energy.

[0008] In order to achieve the above object, the present invention provides a method for constructing bio-ethylene engineering bacteria using carbon dioxide, which is based on chemoautotrophic hydrogen bacteria as the starting strain, and the gene encoding the ethylene-forming enzyme is inserted into the efe The recombinant expression vector is cloned into the expression vector; the recombinant expression vector is integrated into the starting strain to obtain the engineered bacteria.

[0009] Preferably, the above-mentioned chemoautotrophic hydrogen bacteria are selected from H16 ( Cupriavidus necator ), whose deposit number is CGMCC 1.7092.

[0010] Preferably, the above-mentioned gene encoding ethylene-forming enzyme efe The nucleotide sequence is shown in SEQ ID NO.1.

[0011] Preferably, the above expression vector is selected from pBBR1MCS-2.

[0012] The present invention provides a method for constructing an engineered bacterium by transforming a recombinant expression vector into E . coli S17-1 competent cells, the transformed E . coli S17-1 and H16 were mixed and contacted at a volume ratio of 1:5, and conjugation occurred to obtain the engineered bacteria.

[0013] The present invention also provides an engineered bacterium constructed by the above method, which can be used to produce bio-ethylene.

[0014] Preferably, bio-ethylene can be produced by fermenting the above-mentioned engineered bacteria.

[0015] Preferably, the fermentation medium used in the above fermentation comprises the following components: 1 mL / L trace element solution, 2.4 g / L KH2PO4, 2.5 g / L Na2HPO4, 2.0 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 0.1 g / L ammonium ferric citrate, and 0.5 g / L NaHCO3.

[0016] Preferably, the formula of the above-mentioned trace element solution is: 600 mg / L H3BO3, 400 mg / L CoCl2·H2O, 200 mg / L ZnSO4·7H2O, 60 mg / L MnCl2·4H2O, 60 mg / L Na2MoO4·2H2O, 40 mg / L NiCl2·6H2O, and 20 mg / L CuSO4·5H2O.

[0017] The present invention has the following advantages: The present invention provides a method for constructing an engineered bacterium for producing bio-ethylene by utilizing carbon dioxide, and improves the bio-ethylene production by enhancing the carbon dioxide fixation efficiency. efe The engineered bacteria provided by the present invention produce ethylene by using CO2 as the sole carbon source, H2 as the sole energy source, and O2 as the electron acceptor in a mixed gas culture method. C.necator The H16 strain fixes CO2 and catalyzes the metabolic pathway for the synthesis of ethylene, successfully achieving the biocatalytic conversion of CO2 to ethylene, laying the foundation for the development of carbon-neutral, high-value-added synthesis routes based on engineered whole-cell catalytic systems, and achieving synergistic enhancement of carbon fixation and product synthesis.

[0018] This invention utilizes genetic engineering techniques to modify microbial strains, resulting in a novel engineered bacterium. This enriches the resource pool of engineered strains. Furthermore, this engineered bacterium can produce ethylene at room temperature and pressure, making it more environmentally friendly, safer, and faster than traditional ethylene production processes. This method has potential application in the industrial production of ethylene. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the map of the constructed expression plasmid.

[0020] Figure 2 The electrophoresis results of double enzyme digestion identification of the recombinant plasmid containing the ethylene synthase EFE encoding gene.

[0021] Figure 3 The graph shows the results of PCR electrophoresis of the genome of the experimental strain.

[0022] Figure 4 The growth curves of different strains.

[0023] Figure 5 The results of stability analysis of the recombinant plasmid containing the ethylene synthase EFE encoding gene.

[0024] Figure 6 The graph shows the PCR electrophoresis results of serial passages of the experimental strains.

[0025] Figure 7 The electrophoresis results of protein expression in the experimental strains are shown in Figure 2.

[0026] Figure 8 Growth curve under CO2 conditions Figure 9 This is the chromatographic peak diagram for ethylene determination. DETAILED DESCRIPTION

[0027] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0028] Note: Unless otherwise noted, the experimental methods in the following examples are conventional methods, performed according to the techniques and conditions described in literature in the field or according to product specifications. Materials and reagents used in the following examples, unless otherwise noted, are commercially available.

[0029] The present invention provides a method for producing bio-ethylene using CO2, which is achieved by constructing an expression bacterium that can overexpress the gene encoding ethylene-forming enzyme (EFE) and then fermenting the strain, as follows: Among them, the ethylene synthase EFE encoding gene efe The nucleotide sequence (SEQ ID NO.1) is:

[0030] Experimental Example 1 Construction and identification of recombinant plasmid According to the codon preference, the DNA sequence of the suitable hydrogen bacteria host was analyzed and designed, and the 5' end of the ethylene synthase gene sequence Sy-efe was introduced into the Kpn I restriction site and the 3' end was introduced into the EcoR I restriction site. After being synthesized by a biological company, it was cloned into the Kpn I / EcoR I site of the expression vector pBBR1MCS-2 and then sequenced for verification. efe Recombinant plasmids containing genes such as Figure 1 shown.

[0031] The resulting recombinant plasmid was transformed into DH5α via heat shock and stored in glycerol. Simultaneously, the recombinant plasmid was extracted using a kit. The extracted recombinant plasmid was then double-digested with enzymes to verify its identity. The enzyme digestion system is shown in Table 1.

[0032] Table 1 Double enzyme digestion system The enzyme digestion results are as follows Figure 2 As shown in the figure, lane M is the Marker 10000 lane; lane 1 is the lane of the recombinant plasmid enzyme digestion product. It can be seen from the figure that two bands, the vector (about 5000 bp) and the target gene fragment (about 1100 bp), were cut out respectively. Combined with the sequencing results, it shows that the recombinant plasmid was successfully constructed.

[0033] Experimental Example 2 Establishment of recombinant expression strain 1. E. coli S17-1 competent cell culture and transformation 1. Preparation of Competent Cells Remove the E. coli S17-1 strain stored at -80°C, streak it onto LB solid medium, and incubate it at 37°C for 12 hours. Pick a single E. coli S17-1 colony and transfer it to 5 mL of LB liquid medium. Incubate it at 37°C, 200 rpm, shaking overnight. Transfer 1 mL of the bacterial suspension to a conical flask containing 100 mL of LB. Incubate it at 37°C, 200 rpm, shaking for 4-5 hours. Transfer 200 μL of the conical flask to a 96-well plate and monitor the OD600 nm value using a microplate reader until it reaches 0.3-0.5. Place the tube on ice in a foam box. Pre-chill four sterilized 50 mL centrifuge tubes for 10-15 minutes. Keep the conical flask containing 50 mL of 0.1 mM CaCl2 and the test tube containing 10 mL of 0.1 mM CaCl2 and 10% glycerol on ice. Centrifuge the bacterial suspension at 4000 rpm for 10 minutes at 4°C and remove the supernatant to collect the cells. Add 10 mL of pre-chilled 0.1 mM CaCl2 solution to each tube, vortex to mix, and place on ice for 30 minutes. Centrifuge at 4000 rpm for 10 minutes at 4°C and remove the supernatant to collect the cells. Add 2 mL of pre-chilled 0.1 mM CaCl2 and 10% glycerol solution to each tube, vortex to mix, and prepare 20 2 mL EP tubes, each containing 200 mL of the centrifuge tube liquid. Store in a -80°C freezer until needed.

[0034] 2. Heat Shock Transformation Adjust the water bath to 42°C. Thaw two E. coli S17-1 competent cells and recombinant plasmid stored at -80°C in an ice box on a clean bench. Add ice cubes to a foam box and pre-chill a 5mL tube of LB liquid medium. Pipette 50μL of competent cells and 5μL of recombinant plasmid into a sterile 2mL EP tube, vortex to mix, and incubate in a water bath for 30 minutes. Heat shock the tube in a 42°C water bath for 90 seconds, cool on ice for 3 minutes, add 400μL of pre-chilled medium to the EP tube, and gently shake at 37°C for 45 minutes. Centrifuge the culture at 6000 rpm for 5 minutes, remove 250μL of supernatant, vortex to mix, and add 100mL of the culture to an LB plate containing kanamycin. Spread evenly and incubate in an inverted position at 37°C overnight. Pick a single colony from the cultured LB plate containing kanamycin and inoculate it into a 5 mL tube containing LB liquid medium. Incubate the tube for enrichment at 37°C for 12 hours. Transfer 2 mL of the bacterial solution from the tube to a clean, sterile 2 mL EP tube, reserving a portion for culture storage and a portion for plasmid extraction.

[0035] 2. The hydrogen bacteria host selected in this technical solution is Cupriavidus necator H16, whose deposit number is CGMCC1.7092. Take the C. necatorUse an inoculating loop to streak the strain onto a YPM solid plate and incubate overnight. Pick a single colony from the plate, inoculate it into a test tube, and incubate overnight with shaking. Transfer the bacterial suspension to a shake flask. When the colony's OD600 nm value is between 0.5 and 0.7, place the shake flask on ice. Transfer the suspension to a centrifuge tube under a clean bench and centrifuge. Discard the supernatant, add CaCl2 solution, place the tube in an ice bath, and centrifuge. Discard the supernatant, add CaCl2 solution (containing 15% glycerol), resuspend the cells, aliquot into EP tubes, and store in an ultra-low temperature freezer.

[0036] Take C. necator Thaw the competent cells and constructed recombinant plasmid, add ice to a foam box, place the YPM liquid medium test tube inside, aspirate the bacterial solution from the EP tube, add the recombinant plasmid, and place on ice. After heat shock, place on ice, add pre-chilled medium to a new EP tube, and gently shake on a shaker at 30°C. Centrifuge, remove the supernatant, vortex to mix, spread onto a YPM resistance plate containing Kan, and incubate inverted overnight. Pick a single colony from the resistance plate and inoculate into a 5 mL YPM liquid medium test tube for enrichment. Transfer the bacterial solution from the test tube to an EP tube and construct three 25 μL systems for PCR detection of recombinant colonies, reserving a portion for storage.

[0037] 3. The DNA of the recombinant strain was extracted using a bacterial DNA extraction kit, and the target gene was amplified by PCR. The primers used were shown in Table 2, and the PCR reaction was performed using the PCR reaction amplification system configured in Table 3.

[0038] Table 2 Primer sequences Table 3 PCR reaction system for amplifying target genes After PCR was completed, DNA was verified by agarose electrophoresis.

[0039] The ethylene synthase gene was amplified by PCR using the genome of the microbial strain as a template. Figure 3 As shown in the figure, lane M is the marker 10000 lane, and lane T is the lane for the PCR product of the microbial strain genome. It can be seen that the PCR produced a clear band containing the target gene fragment (approximately 1100 bp).

[0040] Experimental Example 3: Strain Activity Test 1. Analysis of recombinant plasmid stability Take the recombinant strain constructed above, streak it with an inoculating loop, and inoculate it onto a solid YPM plate containing Kan. Incubate it upside down overnight. Pick a single colony from the plate and inoculate it into a 5 mL test tube containing YPM culture medium containing Kan. Incubate it with shaking overnight. Every 12 hours, take 1 mL of the bacterial solution and inoculate it into a new 5 mL test tube containing YPM culture medium containing Kan. Incubate it with shaking. Passage it for five generations. Each generation of bacteria is diluted 10-fold with PBS and spread on a non-resistant plate and a Kan-resistant plate. After incubation at 30°C, count the CFU of bacteria on the Kan-resistant plate and the non-resistant plate. Genetic stability is the ratio of the CFU on the resistant plate to the CFU on the non-resistant plate, expressed as a percentage. DNA is extracted from the five-generation blind-transferred strain, and the target fragment is identified by PCR.

[0041] The results are as follows Figure 5 As shown, EP represents the empty plasmid strain and Transformed represents the experimental strain. During five blind passages, the plasmid stability of the recombinant strain gradually increased and then remained stable. The second-generation recombination percentage of the control empty plasmid strain decreased during blind passage, then gradually increased and remained stable. After three blind passages, the plasmid percentage of both the control empty plasmid strain and the recombinant strain remained stable at over 90%.

[0042] The electrophoresis results are as follows Figure 6 As shown, lane M is the Marker 10000 lane; lanes 1-5 are lanes of PCR products after DNA was extracted from the experimental strain after five consecutive generations. It can be seen that in C. necator The presence of the efe gene was detected in the genomic DNA of the engineered bacteria by PCR and gel electrophoresis. The above results indicate that the recombinant plasmid can be used in C. necator The gene was stably inherited in multiple serial subcultures of H16.

[0043] 2. Growth curve determination of different strains Take a recombinant strain (Transformed, hereafter referred to as T), wild-type (WT), or an empty plasmid strain (EP) from a laboratory collection and streak isolated onto a solid YPM plate containing Kan. Incubate the plate upside down overnight. Pick a single colony from the plate and inoculate it into a 5 mL tube containing YPM medium containing Kan. Incubate the plate with shaking overnight. Inoculate 1 mL of the bacterial solution into a 500 mL shake flask containing 100 mL of YPM medium containing Kan and shake. Measure the absorbance of the bacterial solution every 2 hours. Transfer 200 μL of the solution from the conical flask to a 96-well plate. Observe the OD590 value using a microplate reader and record and plot a growth curve.

[0044] The results are as follows Figure 4As shown in the figure, Transformed represents the experimental strain, EP represents the empty plasmid strain, and WT represents the wild-type strain. All strains exhibited similar changes. Within the first 4 hours, all strains were in the lag phase, then entered the logarithmic growth phase with rapid growth. After 28 hours, the growth rate decreased, gradually dropping to zero, and then entered the stationary phase and decay phase. Throughout the growth process, the growth rate of the WT group was slightly higher than that of the EP and T groups, but no significant differences were observed, indicating that the recombinant plasmid had no significant effect on cell growth.

[0045] 3. SDS-PAGE gel electrophoresis Laboratory-collected engineered bacterial strains (Transformed, hereinafter referred to as T), wild-type bacteria (WT), and an empty plasmid strain (EP) were streaked onto solid YPM plates containing Kan using an inoculating loop and incubated inverted overnight. A single colony was picked from the plate and inoculated into a 5 mL test tube containing YPM medium containing Kan and incubated overnight with shaking. One mL of the bacterial culture was inoculated into a 500 mL shake flask containing 100 mL of YPM medium containing Kan and incubated for approximately 6–8 hours. When the colony OD600nm value reached 0.6–0.8, the engineered bacteria were divided into three aliquots, each receiving 0.1 mM IPTG and 0.5 mM lactose (Lac), while the other aliquot remained uninduced. The cells were incubated overnight to induce protein expression. The wild-type strain and the empty plasmid strain served as controls. The overnight culture was centrifuged at 8000 rpm for 5 minutes, washed with pre-chilled PBS, resuspended in PBS, and disrupted by sonication in an ice-cold water mixture. The disrupted bacterial solution was centrifuged, 60 μL of the supernatant was added to 20 μL of 4x protein loading buffer, mixed well, and boiled in boiling water for 10 minutes for protein loading. According to the instructions of the Solebol SDS-PAGE gel preparation kit, 12% separation gel and 5% stacking gel were prepared for SDS-PAGE analysis. The results are shown in Figure 7 shown.

[0046] Experimental Example 4 Determination of growth activity of recombinant strains under CO2 conditions Single colonies of wild-type (WT), empty plasmid (EP), and engineered (Transformed) strains were inoculated into 5 mL YPM tubes containing the corresponding resistance strains and cultured at 30°C, 150 rpm, and shaking for 12–16 hours. The cells were harvested by centrifugation at 8000 rpm for 5 minutes, the supernatant discarded, and the cells were washed and resuspended in an equal volume of inorganic salt medium. A 5% inoculum (v / v) was transferred to a 1 L, pressure-resistant, thick-walled vented bottle containing 100 mL of culture medium. Sterile mixed gas was continuously aerated until the exhaust gas detection was stable. The system was sealed and incubated in a constant-temperature shaker at 30°C, 150 rpm. Aeration was repeated every 24 hours to replace the culture ambient atmosphere, and samples were collected for growth curve monitoring.

[0047] The autotrophic growth curve was constructed by regularly measuring the OD600 values ​​of WT, EP and Transformed in gas culture to evaluate the effect of the recombinant plasmid on bacterial proliferation in an autotrophic environment. Figure 8 As shown, the growth dynamics of the three strains exhibited similar trends. Throughout the culture period, all strains maintained a continuous proliferation state. In the gas culture system, although the WT strain proliferated slightly faster than the transformed strain, the two strains maintained the same growth trend, indicating that the recombinant plasmid carried by the transformed strain did not significantly interfere with its normal physiological and metabolic activities.

[0048] Experimental Example 5 Determination of ethylene production by recombinant strains Take the recombinant engineered bacterial strain constructed above and streak it onto a solid YPM plate containing Kan using an inoculating loop. Mark the plate and invert it to incubate overnight at 30°C in a 30°C incubator. Pick a single colony from the plate and inoculate it into a 5mL test tube containing YPM medium containing Kan. Incubate the tube with shaking at 30°C and 200 rpm overnight. Inoculate 1mL of the bacterial solution into a 500mL shake flask containing 100mL of YPM medium containing Kan. Incubate the tube at 30°C and 200 rpm overnight.

[0049] The YPM culture medium used in the fermentation process contains 1 mL / L of trace element solution, 2.4 g / L KH2PO4, 2.5 g / L Na2HPO4, 2.0 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 0.1 g / L ammonium ferric citrate, and 0.5 g / L NaHCO3. The trace element solution contains 600 mg / L H3BO3, 400 mg / L CoCl2·H2O, 200 mg / L ZnSO4·7H2O, 60 mg / L MnCl2·4H2O, 60 mg / L Na2MoO4·2H2O, 40 mg / L NiCl2·6H2O, and 20 mg / L CuSO4·5H2O.

[0050] The headspace above the conical flask was extracted with a gas-tight syringe and injected into GC-MS for analysis.

[0051] Ethylene determination results Figure 9 Figure 1 shows the chromatogram of the ethylene standard, where A is the chromatogram of the sample, and C is the chromatogram of the wild-type strain. It can be seen that the retention time of the sample is close to that of the ethylene standard, at around 1.50 min. There is no obvious peak marker in the headspace gas of the wild-type strain, which further indicates that the hydrogen bacteria C. necator The new carbon fixation pathway has been successfully constructed and can produce bio-ethylene.

[0052] In summary, the recombinant expression bacteria constructed by the present invention can be used to produce bio-ethylene, providing a new carbon fixation pathway that can effectively reduce greenhouse gas concentrations and alleviate global warming. The generated ethylene can also realize the resource utilization of waste, and has significant practical value.

[0053] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for constructing bio-ethylene production engineering bacteria using carbon dioxide, characterized in that: Using chemoautotrophic hydrogen bacteria as the starting strain, the gene encoding ethylene-forming enzyme efe The recombinant expression vector is cloned into the expression vector; the recombinant expression vector is integrated into the starting strain to obtain the engineered bacteria.

2. The method according to claim 1, wherein: The chemoautotrophic hydrogen bacteria are selected from H16 ( Cupriavidus necator ), whose deposit number is CGMCC 1.7092.

3. The method according to claim 1, wherein: The gene encoding ethylene-forming enzyme efe The nucleotide sequence is shown in SEQ ID NO.

1.

4. The method according to claim 1, wherein: The expression vector is selected from pBBR1MCS-2.

5. The method according to any one of claims 1 to 4, characterized in that By transforming the recombinant expression vector into E . coli S17-1 competent cells, the transformed E . coli S17-1 and H16 were mixed and contacted at a volume ratio of 1:5, and conjugation occurred to obtain the engineered bacteria.

6. An engineered bacterium constructed by the method according to any one of claims 1 to 5.

7. Use of the engineered bacteria according to claim 6 in the production of bio-ethylene.

8. The use according to claim 7, characterized in that Bio-ethylene is produced by fermenting the engineered bacteria as claimed in claim 6.

9. The use according to claim 8, characterized in that The fermentation medium used in the fermentation comprises the following components: 1 mL / L trace element solution, 2.4 g / L KH2PO4, 2.5 g / L Na2HPO4, 2.0 g / L (NH4)2SO4, 0.5 g / LMgSO4·7H2O, 0.1 g / L ammonium ferric citrate, and 0.5 g / L NaHCO3.

10. The use according to claim 9, characterized in that The formula of the trace element solution is: 600 mg / LH3BO3, 400 mg / L CoCl2·H2O, 200 mg / L ZnSO4·7H2O, 60 mg / L MnCl2·4H2O, 60 mg / LNa2MoO4·2H2O, 40 mg / L NiCl2·6H2O, and 20 mg / L CuSO4·5H2O.

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