Composition for leaching lithium, nickel or cobalt using a Chlorella vulgaris strain, and method for leaching lithium, nickel or cobalt

Abstract

The present invention relates to a composition for leaching lithium, nickel, or cobalt using a Chlorella vulgaris strain and a method for leaching lithium, nickel, or cobalt.

Description

[Technical Field]

[0001] This patent application claims priority based on Korean Patent Application No. 10-2023-0009737, filed with the Korean Intellectual Property Office on January 25, 2023, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for leaching lithium, nickel, or cobalt using a Chlorella vulgaris strain, and a method for leaching lithium, nickel, or cobalt. [Background technology]

[0003] Chlorella vulgaris is a unicellular microalgae belonging to the Chlorophyta phylum. Chlorella vulgaris possesses photosynthetic mechanisms and can grow rapidly using only light, carbon dioxide, water, and small amounts of minerals. Therefore, it is widely used in research on the mass production of useful substances (photobioreactors).

[0004] In addition, because the lipid content per biomass is relatively high at about 42%, it is often used in research and development of biofuels that can be used as an alternative to biodiesel. 90 Research has also reported that Sr can be removed.

[0005] Electroporation was performed using selection markers such as the hygromycin B resistance gene, the zeocin resistance gene, and the chloramphenicol acetyltransferase gene (CAT gene). Integration into chromosomal DNA was confirmed, but the transformants were only maintained temporarily and lost resistance within a short period of time.

[0006] Transformation methods include glass beads, electroporation, and Agrobacterium-mediated transformation. However, the use of Chlorella vulgaris remains limited due to the lack of efficient selectable markers and transformation systems.

[0007] With technological advances, electricity usage is increasing rapidly, and the efficiency of energy consumption and production is also improving. Given the rapid increase in electricity demand and the corresponding production efficiency, the world is turning its attention to nuclear power generation. However, alongside this high production efficiency, there are always concerns about the risks of nuclear accidents and contamination by radioactive and metal ions in contaminated water, and these issues remain unresolved.

[0008] Furthermore, Japan faces a national problem of being dependent on imports of rare metals used in industry, such as Sr, Cs, Li, Ni, and Co. This problem has become even more pronounced with the expansion of the secondary battery market, and the issue of recycling these rare metals has emerged as a major problem both industrially and nationally.

[0009] Hydrometallurgy and pyrometallurgy are primarily used to recover metals from raw ores and secondary resources (such as waste catalysts and electronic devices). However, hydrometallurgy using acid and alkali leaching methods poses serious environmental pollution problems because strong acids and strong alkali solvents are used to leach metals. Furthermore, pyrometallurgy generates large amounts of sulfide gas, posing numerous challenges in terms of facility operation. Given these circumstances, interest is growing in bioleaching as a leaching method with a lower environmental impact.

[0010] Bioleaching is a type of hydrometallurgical process that uses microorganisms capable of leaching metals to recover valuable metals. However, bioleaching, which is considered environmentally friendly, has the drawback of low metal leaching efficiency. Therefore, there is currently a strong demand for the development of bioleaching technology that is highly efficient in metal leaching and has a low environmental impact. Summary of the Invention [Problem to be solved by the invention]

[0011] The present inventors have confirmed that lithium, nickel, or cobalt can be leached with high efficiency from materials containing lithium, nickel, or cobalt using Chlorella vulgaris strains. In particular, they have confirmed that Chlorella vulgaris strains transformed with the vectors of the present invention exhibit superior lithium, nickel, or cobalt leaching efficiency, leading to the completion of the present invention.

[0012] We predicted the promoter and terminator sequences of Coccomyxa C-169 (hereafter referred to as C-169; previously known as Chlorella vulgaris, but renamed), obtained the sequence, and synthesized a fusion with the Sh ble gene. No introns were inserted into the Streptomyces verticillus bleomycin (Sh ble) gene. The synthesized DNA fragment was digested with SwaI / KpnI restriction enzymes and cloned into the pSP124S vector, which was named pKA650.

[0013] In addition, the target gene, beta-type carbonic anhydrase, was cloned into the middle of the Streptomyces verticillus bleomycin (Sh ble) gene in the pKa650 vector, and the resulting vector was named pJG002.

[0014] The transformants were then transformed by gold particle bombardment, and selectively cultured in a medium containing Zeocin. Transformants were produced by obtaining colonies through selective culture.

[0015] We also confirmed that lithium, nickel, or cobalt can be leached with high efficiency from materials containing lithium, nickel, or cobalt using Chlorella vulgaris or its transformants.

[0016] Therefore, an object of the present invention is to provide a composition for leaching lithium, nickel, or cobalt, which comprises one or more members selected from the group consisting of a Chlorella vulgaris strain, a culture of the strain, a concentrate of the culture, and a dried product of the culture.

[0017] Another object of the present invention is to provide a method for leaching lithium, nickel, or cobalt by bioleaching, which includes a mixing step of mixing one or more selected from the group consisting of a Chlorella vulgaris strain, a culture of the strain, a concentrate of the culture, and a dried product of the culture with a substance containing lithium, nickel, or cobalt, and a leaching step of leaching lithium, nickel, or cobalt for a predetermined period of time.

[0018] Another object of the present invention is to provide a use of Chlorella vulgaris strain for lithium, nickel or cobalt leaching. [Means for solving the problem]

[0019] The present invention relates to a composition for leaching lithium, nickel, or cobalt using a Chlorella vulgaris strain, and a method for leaching lithium, nickel, or cobalt.

[0020] The present invention will now be described in more detail.

[0021] One aspect of the present invention relates to a composition for leaching lithium, nickel, or cobalt, comprising at least one selected from the group consisting of a Chlorella vulgaris strain, a culture of the strain, a concentrate of the culture, and a dried product of the culture.

[0022] In the present invention, the Chlorella vulgaris strain can leach lithium, nickel, or cobalt by crystallizing lithium, nickel, or cobalt from a substance containing lithium, nickel, or cobalt through biomineralization.

[0023] As used herein, the term "leaching" refers to the separation of a target component and can be used interchangeably with "separation," "extraction," or "elution."

[0024] As used herein, the term "leaching composition" refers to a composition intended for the separation of a target component, and may be used interchangeably with "biomineralization composition" or "bioleaching composition."

[0025] In the present invention, the strain may be transformed with a vector comprising: Coccomyxa C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo) small subunit 2 (rbcS2) gene promoter; beta-type carbonic anhydrase-coding nucleotide sequence of Coccomyxa subellipsoidea C-169; and Terminator sequence of the rbcS2 gene of Coccomyxa C-169.

[0026] In the present invention, the promoter may contain the nucleotide sequence of SEQ ID NO: 2, or may contain a nucleotide sequence substantially identical to the nucleotide sequence of SEQ ID NO: 2. For example, the promoter may consist of the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

[0027] In one example of the present invention, a promoter containing the base sequence of SEQ ID NO: 2 may be a sequence that further contains 1 to 50 bp of bases at the 3' end of the sequence of SEQ ID NO: 2 contained in SEQ ID NO: 1, but is not limited to this.

[0028] In the present invention, the terminator sequence may include the base sequence of SEQ ID NO: 3, or may include a base sequence that is substantially identical to the base sequence of SEQ ID NO: 3, for example, it may consist of the base sequence of SEQ ID NO: 3, but is not limited to this.

[0029] In the present invention, the protein-encoding nucleotide sequence of interest may be operatively linked to a promoter.

[0030] In the present invention, "operatively linked" refers to a functional connection between an expression control sequence of a nucleic acid (e.g., a promoter sequence, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, which enables the control sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0031] In the present invention, the nucleotide sequence encoding the target protein may be linked to the 3' end of the terminator sequence, but is not limited thereto.

[0032] In the present invention, the vector may further comprise a selection marker.

[0033] In the present invention, the selection marker may be, but is not limited to, an antibiotic resistance gene.

[0034] In the present invention, the antibiotic may be one or more selected from the group consisting of spectinomycin, paromomycin, ampicillin, zeocin, and bleomycin, but is not limited thereto.

[0035] In the present invention, various selectable marker genes for antibiotics currently in use can be selected, such as aminoglycoside phosphotransferase, which confers resistance to kanamycin antibiotics, and chloramphenicol acetyltransferase, which is involved in resistance to chloramphenicol antibiotics.

[0036] In the present invention, when the selection marker is bleomycin, the selection marker may contain the base sequence of SEQ ID NO: 4, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 4, for example, may consist of the base sequence of SEQ ID NO: 4, but is not limited to this.

[0037] In the present invention, the method for selecting transformed Chlorella vulgaris into which a selection marker has been introduced can be easily carried out by a method well known to those skilled in the art using the phenotype expressed by the selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0038] In the present invention, the vector may further comprise a gene encoding a reporter molecule.

[0039] In the present invention, the reporter molecule may be one or more selected from the group consisting of growth-promoting proteins, fluorescent proteins and hydrolases, but is not limited thereto.

[0040] In the present invention, the fluorescent protein may be, but is not limited to, luciferase.

[0041] In the present invention, the hydrolase may be, but is not limited to, β-glucuronidase.

[0042] In the present invention, the vector may contain the base sequence of SEQ ID NO: 1, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 1, for example, the vector may consist of the base sequence of SEQ ID NO: 1, but is not limited to this.

[0043] In the present invention, the vector may be for transformation of Chlorella vulgaris using gold particle bombardment.

[0044] In the present invention, the term "vector" refers to a means for expressing a gene of interest in a host cell, and includes, for example, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector such as an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector.

[0045] In the present invention, vectors that can be used as recombinant vectors may be prepared by manipulating plasmids frequently used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, the pGEX series, the pET series, and pUC19), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), or viruses (e.g., SV40), and may be, for example, but not limited to, the pSP124S vector backbone.

[0046] In the present invention, a vector can typically be constructed as a vector for cloning or a vector for expression.

[0047] In the present invention, vectors for expression may be those commonly used in the art for expressing target proteins in plants, animals or microorganisms.

[0048] In the present invention, vectors can be constructed by various methods known in the art.

[0049] In the present invention, the term "substantial identity" means that, when each base sequence is aligned with any other base sequence to maximize correspondence and the sequences are analyzed, the any other base sequence has sequence homology of 70% or more, 90% or more, or 98% or more with the respective base sequence.

[0050] Another aspect of the present invention relates to a method for leaching lithium, nickel, or cobalt by bioleaching, which includes a mixing step of mixing one or more selected from the group consisting of a Chlorella vulgaris strain, a culture of the strain, a concentrate of the culture, and a dried product of the culture with a substance containing lithium, nickel, or cobalt, and a leaching step of leaching lithium, nickel, or cobalt for a predetermined period of time.

[0051] In the present invention, the lithium, nickel or cobalt leaching method comprises the following transformation step before the mixing step, and the Chlorella vulgaris strain in the mixing step may be a transformed Chlorella vulgaris strain: The transformation step involves introducing a vector containing the promoter of the Coccomyxa C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo) small subunit 2 (rbcS2) gene, the target protein-coding nucleotide sequence, and the terminator sequence of the Coccomyxa C-169 rbcS2 gene into Chlorella vulgaris.

[0052] In the present invention, the promoter may contain the base sequence of SEQ ID NO: 2, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 2, for example, may consist of the base sequence of SEQ ID NO: 2, but is not limited thereto.

[0053] In one example of the present invention, a promoter containing the base sequence of SEQ ID NO: 2 may be a sequence that further contains 1 to 50 bp of bases at the 3' end of the sequence of SEQ ID NO: 2 contained in SEQ ID NO: 1, but is not limited to this.

[0054] In one example of the present invention, a promoter containing the base sequence of SEQ ID NO: 2 may be a sequence that further contains 1 to 50 bp of bases at the 3' end of the sequence of SEQ ID NO: 2 contained in SEQ ID NO: 7, but is not limited to this.

[0055] In the present invention, the terminator sequence may include the base sequence of SEQ ID NO: 3, or may include a base sequence that is substantially identical to the base sequence of SEQ ID NO: 3, for example, it may consist of the base sequence of SEQ ID NO: 3, but is not limited to this.

[0056] In the present invention, the protein-encoding nucleotide sequence of interest may be operatively linked to a promoter.

[0057] In the present invention, the nucleotide sequence encoding the target protein may be linked to the 3' end of the terminator sequence, but is not limited thereto.

[0058] In the present invention, the vector may further comprise a selection marker.

[0059] In the present invention, the selection marker may be, but is not limited to, an antibiotic resistance gene.

[0060] In the present invention, the antibiotic may be one or more selected from the group consisting of spectinomycin, paromomycin, ampicillin, zeocin, and bleomycin, but is not limited thereto.

[0061] In the present invention, various selectable marker genes for antibiotics currently in use can be selected, such as aminoglycoside phosphotransferase, which confers resistance to kanamycin antibiotics, and chloramphenicol acetyltransferase, which is involved in resistance to chloramphenicol antibiotics.

[0062] In the present invention, when the selection marker is bleomycin, the selection marker may contain the base sequence of SEQ ID NO: 4, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 4, for example, may consist of the base sequence of SEQ ID NO: 4, but is not limited to this.

[0063] In the present invention, the method for selecting transformed Chlorella vulgaris into which a selection marker has been introduced can be easily carried out by a method well known to those skilled in the art using the phenotype expressed by the selection marker. For example, if the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

[0064] In the present invention, the vector may further comprise a gene encoding a reporter molecule.

[0065] In the present invention, the reporter molecule may be one or more selected from the group consisting of growth-promoting proteins, fluorescent proteins and hydrolases, but is not limited thereto.

[0066] In the present invention, the fluorescent protein may be, but is not limited to, luciferase.

[0067] In the present invention, the hydrolase may be, but is not limited to, β-glucuronidase.

[0068] In the present invention, the vector may contain the base sequence of SEQ ID NO: 1, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 1, for example, the vector may consist of the base sequence of SEQ ID NO: 1, but is not limited to this.

[0069] In the present invention, the vector may contain the base sequence of SEQ ID NO: 7, or may contain a base sequence that is substantially identical to the base sequence of SEQ ID NO: 7, for example, the vector may consist of the base sequence of SEQ ID NO: 7, but is not limited to this.

[0070] In the present invention, the vector may be for transformation of Chlorella vulgaris using gold particle bombardment.

[0071] In the present invention, vectors that can be used as recombinant vectors may be prepared by manipulating plasmids frequently used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, the pGEX series, the pET series, and pUC19), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), or viruses (e.g., SV40), and may be, for example, but not limited to, the pSP124S vector backbone.

[0072] In the present invention, a vector can typically be constructed as a vector for cloning or a vector for expression.

[0073] In the present invention, vectors for expression may be any of those commonly used in the art for expressing target proteins in plants, animals or microorganisms.

[0074] In the present invention, vectors can be constructed by various methods known in the art.

[0075] In the present invention, the transformation step may be carried out using gold particles bombardment.

[0076] In the present invention, the cell stage of the transformation step may be the log phase, which is most efficient.

[0077] In the present invention, the log phase may have an OD686 value of 0.4 to 0.6, 0.45 to 0.6, 0.5 to 0.6, or 0.55 to 0.6, for example, 0.6.

[0078] In the present invention, the cell density at the transformation stage is 5.0*10 6 ~8.0*10 7 per 60mm Diameter, 1.0*10 7 ~8.0*10 7 per 60mm diameter, 2.0*10 7 ~8.0*10 7 per 60mm diameter, 3.0*10 7 ~8.0*10 7 per 60mm diameter, 4.0*10 7 ~8.0*10 7 per 60mm diameter, 1.0*10 7 ~7.0*10 7 per 60mm diameter, 2.0*10 7 ~7.0*10 7 per 60mm diameter, 3.0*10 7 ~7.0*10 7 per 60mm diameter, 4.0*10 7 ~7.0*10 7 per 60mm diameter, 1.0*10 7 ~6.0*10 7 per 60mm diameter, 2.0*10 7~6.0*10 7 per 60mm diameter, 3.0*10 7 ~6.0*10 7 per 60mm diameter, 4.0*10 7 ~6.0*10 7 per 60mm diameter, 1.0*10 7 ~5.0*10 7 per 60mm diameter, 2.0*10 7 ~5.0*10 7 per 60mm diameter, 3.0*10 7 ~5.0*10 7 per 60mm diameter, 4.0*10 7 ~5.0*10 7 per 60mm diameter, e.g., 4.8*10 7 It may be per 60mm diameter.

[0079] In the present invention, the vacuum in the transformation step may be 27.0 to 29.0 inches Hg, 27.5 to 29.0 inches Hg, 28.0 to 29.0 inches Hg, or 28.5 to 29.0 inches Hg, for example, 29.0 inches Hg.

[0080] In the present invention, the target distance in the transformation step may be 3 to 9, for example, 3, 6 or 9.

[0081] In the present invention, the pressure in the transformation step may be 1200 to 1300 psi, 1210 to 1300 psi, 1220 to 1300 psi, 1230 to 1300 psi, 1240 to 1300 psi, 1250 to 1300 psi, 1260 to 1300 psi, 1270 to 1300 psi, 1280 to 1300 psi, 1290 to 1300 psi, for example, 1300 psi.

[0082] In the present invention, the substance containing lithium, nickel or cobalt may be one or more selected from the group consisting of LiCl solution, Li2SO4 solution, a battery containing lithium, NiCl2 solution, NiSO4 solution, a battery containing nickel, CoCl2 solution, CoSO4 solution, a battery containing cobalt, waste batteries, wastewater and salt lakes, but is not limited thereto. [Effects of the Invention]

[0083] The present invention relates to a composition for leaching lithium, nickel, or cobalt using a Chlorella vulgaris strain, and a method for leaching lithium, nickel, or cobalt. [Brief explanation of the drawings]

[0084] [Figure 1] 1 is a pKA650 vector map according to one embodiment of the present invention. [Figure 2] 1 is a photograph showing the results of confirming that transformation efficiency was low and transformation was insufficient in an example of the present invention using the glass bead method. [Figure 3] 1 is a photograph showing the results of introducing a resistance gene using a vector system according to an embodiment of the present invention and obtaining a mutant having antibiotic resistance through transformation therewith. [Figure 4] 1 is a photograph showing the results of confirming the genetic information of a mutant obtained according to an embodiment of the present invention. [Figure 5] 1 is a photograph showing the results of subculture to confirm the maintenance of a mutant according to one embodiment of the present invention. [Figure 6] 1 is a map of the pJG002 vector according to one embodiment of the present invention. [Figure 7] According to one embodiment of the present invention, biolistic transformation was carried out in the same manner as in Example 2, and the results show that antibiotic resistance was maintained even after subculture at each concentration. [Figure 8]FIG. 1 is a photograph showing the results of confirming the band size of approximately 43 kDa of the target protein, beta-type carbonic anhydrase, by whole protein SDS-PAGE to confirm gene and protein expression in a transformant according to one embodiment of the present invention. [Figure 9] 1 shows the results of MALDI-TOF performed to confirm the band of a target protein according to one embodiment of the present invention. [Figure 10] 1 is a photograph showing the results of confirming the presence or absence of a target gene by Southern blot for genetic confirmation according to one embodiment of the present invention. [Figure 11] 1 is a photograph showing the results of a comparison of Sr biomineralization between a wild type and a transformant according to one example of the present invention. [Figure 12] 1 shows the results of an ICP-MS analysis for quantitative comparison of Sr biomineralization between a wild type and a transformant according to one embodiment of the present invention. [Figure 13] 1 is a graph showing the results of confirming the removal rate depending on a single LiCl concentration according to an embodiment of the present invention. [Figure 14] 1 is a graph showing the results of confirming the removal rate depending on a single LiCl concentration according to an embodiment of the present invention. [Figure 15] 1 is a graph showing the results of measuring the effects of sodium and sulfate (by concentration: 300, 600, 900 mM) according to an embodiment of the present invention. [Figure 16] 1 is a graph showing the results of measuring the effects of sodium and sulfate (by concentration: 300, 600, 900 mM) according to an embodiment of the present invention. [Figure 17] 1 is a graph showing the composition of a lithium battery solution according to an embodiment of the present invention. [Figure 18] 1 is a graph showing the results of Li crystallization in a lithium battery solution according to one embodiment of the present invention. [Figure 19]1 is a graph showing the results of crystallization with Na and S (300, 600, and 900 mM, respectively) in a lithium battery solution according to one embodiment of the present invention. [Figure 20] 1 is a graph showing the results of crystallization with Na and S (300, 600, and 900 mM, respectively) in a lithium battery solution according to one embodiment of the present invention. [Figure 21] 1 is a graph showing the results of confirming the removal rate as a function of a single NiCl2 concentration according to an embodiment of the present invention. [Figure 22] 1 is a graph showing the results of confirming the removal rate as a function of a single NiCl2 concentration according to an embodiment of the present invention. [Figure 23] 1 is a graph showing the results of measuring the effects of sodium and sulfate (by concentration: 300, 600, 900 mM) according to an embodiment of the present invention. [Figure 24] 1 is a graph showing the results of measuring the effects of sodium and sulfate (by concentration: 300, 600, 900 mM) according to an embodiment of the present invention. [Figure 25] 2 is a graph showing the composition of a nickel battery solution according to an embodiment of the present invention. [Figure 26] 1 is a graph showing the results of Ni crystallization in a nickel battery solution according to one embodiment of the present invention. [Figure 27] 1 is a graph showing the results of crystallization with Na and S (300, 600, and 900 mM, respectively) in a nickel battery solution according to one embodiment of the present invention. [Figure 28] 1 is a graph showing the results of crystallization with Na and S (300, 600, and 900 mM, respectively) in a nickel battery solution according to one embodiment of the present invention. [Figure 29] 1 is a graph showing the results of confirming the removal rate according to a single CoCl2 concentration according to an embodiment of the present invention. [Figure 30] 1 is a graph showing the results of confirming the removal rate according to a single CoCl2 concentration according to an embodiment of the present invention. [Figure 31]1 is a graph showing the results of measuring the effects of sodium and sulfate (by concentration: 300, 600, 900 mM) according to an embodiment of the present invention. [Figure 32] 1 is a graph showing the results of measuring the effects of sodium and sulfate (by concentration: 300, 600, 900 mM) according to an embodiment of the present invention. [Figure 33] 1 is a graph showing the composition of a cobalt battery solution according to an embodiment of the present invention. [Figure 34] 1 is a graph showing the crystallization results of Co in a cobalt battery solution according to one embodiment of the present invention. [Figure 35] 1 is a graph showing the results of crystallization with Na and S (300, 600, and 900 mM, respectively) in a cobalt battery solution according to one embodiment of the present invention. [Figure 36] 1 is a graph showing the results of crystallization with Na and S (300, 600, and 900 mM, respectively) in a cobalt battery solution according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0085] A composition for leaching lithium, nickel, or cobalt, comprising one or more members selected from the group consisting of a Chlorella vulgaris strain, a culture of said strain, a concentrate of said culture, and a dried product of said culture.

[0086] (Mode for Carrying Out the Invention) The present invention will be described in more detail below with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0087] Preparation Example 1. Plasmid construction and cloning The sequence of the bleomycin resistance gene (Sh ble) was obtained from the genomic DNA of Streptomyces verticillus. The promoter sequence of the Coccomyxa C-169 rbcS2 gene was joined to the 5' end of the bleomycin resistance gene, and the terminator sequence of the Coccomyxa C-169 rbcS2 gene was joined to the 3' end to synthesize the entire sequence, Sh ble (GenScript, USA). The synthesized gene was digested with SwaI and KpnI restriction enzymes and inserted into the pSP124S vector. The resulting plasmid was designated pKA650, and its vector map is shown in Figure 1.

[0088] As can be seen in Figure 1, OriV in the vector map is the replication origin, Sh ble is the Sh ble gene bound to the Streptomyces verticillus bleomycin promoter and terminator, AmpR is the ampicillin resistance gene, C-169-ble is the promoter sequence of the Coccomyxa C-169 rbcS2 gene, and the 3' portion is the terminator portion of the Coccomyxa C-169 rbcS2 gene.

[0089] Preparation Example 2: Transformation into Chlorella vulgaris

[0090] 2-1. Transformation using the glass bead method A simple and rapid glass bead transformation method was used, in which glass beads are inserted and physical force is applied, creating holes in the cell membrane due to physical friction with the cells, allowing the introduction of the target plasmid.

[0091] The experiment was performed under the conditions listed in Table 1. Specifically, cells were vortexed using glass beads at room temperature (25°C) to partially disrupt the cells. The beads and supernatant were then separated by gravity, and DNA was added to the supernatant. The cells were then allowed to recover by slow rotation at 37°C. After dispensing onto solid medium containing antibiotics, transformants were confirmed. The results are shown in Figure 2.

[0092] [Table 1]

[0093] As can be seen from FIG. 2, the efficiency was low and transformation was not sufficient.

[0094] 2-2. Transformation using the gene gun method Transformation was performed using gold particle bombardment with a gene gun, a method of plant transformation. This technique is also called particle acceleration or biolistics, but the official name of the device called a gene gun is microparticle bombardment. Transformation is achieved by coating a plasmid with microparticles. Microparticles are very heavy compared to their size, allowing them to penetrate cells effectively. Microparticles are fired at cells at high speed while a steel net is placed around them, ensuring that more particles reach the cells. DNA coated on the microparticles that enter the cells is released and can be incorporated into the plant's genome. Transformation was performed using gold as the microparticles.

[0095] Specifically, experiments were conducted while changing various conditions to perform transformation using gold particle bombardment, as shown in Table 2 below. First, the vacuum and helium pressure were fixed, and the experiment was carried out while taking into consideration the cell stage and concentration, gold particle injection range, target distance, and experimental environment of the cells.

[0096] In the process of establishing the conditions, experiments were conducted at 47mm, 50mm, and 60mm, taking into consideration the injection range and pressure of gold particles from the gene gun. After examining the Target Distance as the distance changed, experiments were conducted at Target Distance 3 as the optimum position. Furthermore, since the membrane of the microalgae becomes thicker as the cell stage progresses too far, making experiments difficult, experiments were conducted from the early stages according to changes in OD value. Furthermore, experiments were conducted by adjusting the cell density according to the diameter.

[0097] [Table 2]

[0098] As can be seen from Table 2 and Figure 3, in unsuccessful cases, growth was not possible on solid medium containing antibiotics, whereas in successful cases, colonies were formed, but promoter operation (mutants) was confirmed only under conditions 160704. Subsequently, the target protein (carbonic anhydrase) was introduced under the same conditions to form mutants.

[0099] Experimental example 1. Confirmation of genetic information To confirm the genetic information, genomic DNA was isolated and the introduced gene was confirmed by PCR and DNA sequencing. Specifically, the primer set shown in Table 3 below was prepared for PCR to confirm the gene. Using this primer set, pre-denaturation was performed at 98°C for 8 minutes, followed by 30 cycles of 98°C for 1 minute, 53.5°C for 30 seconds, and 72°C for 1 minute, followed by PCR for 7 minutes at 72°C. This was then confirmed by sequencing analysis. The results are shown in Table 1.

[0100] To confirm Sh ble, the entire sequence of the bleomycin resistance gene of interest, obtained by splicing the promoter sequence of the Coccomyxa C-169 rbcS2 gene at the 5' end and the terminator sequence of the Coccomyxa C-169 rbcS2 gene at the 3' end, we performed PCR using the M13 primer set. Pre-denaturation was performed at 98°C for 8 minutes, followed by 30 cycles of 98°C for 1 minute, 54°C for 40 seconds, and 72°C for 2 minutes and 30 seconds. PCR was then performed at 72°C for 1 minute. The target DNA band was confirmed by PCR, and the results are shown in Figure 4.

[0101] [Table 3]

[0102] As can be seen in Figure 4, a band was confirmed at approximately 2 kb, which is the full length of the target gene. DNA gene analysis was performed on this to confirm whether it matched the target gene, and the results are shown in Table 4.

[0103] [Table 4]

[0104] As can be seen in Table 4, multiple sequence alignment using CLUSTAL 2.1 confirmed that the DNA base sequence of the target gene, Sh-ble, was a perfect match with the obtained gene information.

[0105] Experimental Example 2: Confirmation of mutant maintenance Subculture was performed to confirm the maintenance of the mutant, and the results are shown in Figure 5. As can be seen from Figure 5, the maintenance of the mutant was confirmed by subculture.

[0106] Experimental Example 3: Biomineralization

[0107] Example 1: Plasmid construction and cloning This study involved constructing and cloning a plasmid for expressing a target protein using the pKA650 vector, conferring zeocin resistance and cloning. The bleomycin resistance gene (Sh ble) sequence was obtained from the genomic DNA of Streptomyces verticillus (Sh ble). The promoter and terminator sequences of the Coccomyxa C-169 rbcS2 (ribulose-1,5-bisphosphate carboxylase / oxygenase small subunit) gene were ligated to the 5' and 3' ends of Sh ble, respectively, to synthesize the entire sequence. This sequence, designated pKA650, was synthesized by replacing the Sh ble intermediate gene, BleoR, with the C-169 beta-type carbonic anhydrase gene, and then inserting the overlapping promoter portion using the restriction enzymes Kpn I and Apa I. The cloned plasmid is shown in Figure 6 and was designated pJG002.

[0108] Example 2: Transformation of Chlorella vulgaris The transformation method using the pJG002 vector is the same as the transformation method using the pKA650 vector.

[0109] Specifically, to prepare competent cells, 300 ml of sterilized MBM medium (KNO3 2.5 mM, MgSO4 7H2O 0.3 mM, K2HPO4 0.43 mM, KH2PO4 1.29 mM, NaCl 0.43 mM, CaCl2 * 2H2O 0.068 mM, FeSO4 * 7H2O 0.1 g, A5 metal mix ml / L) was prepared in a 500 ml flask, inoculated with Chlorella vulgaris, and cultured at 23°C and 100 rpm. 686When the value reaches 0.5 to 0.6, the Chlorella vulgaris is collected by centrifugation and the cell count is then confirmed.

[0110] After checking the cell count, the cell density was set at 4.8*10 7 Next, for gold particle bombardment, the pKA650 to be introduced is linearized with the restriction enzyme KpnI. The retaining cap, brass adjustable nest, microcarrier holder, stopping screen, and macrocarrier, all prepared in a sterile environment, are prepared, and the rupture disk is washed with 70% isopropanol. While the rupture disk is drying, the plasmid and gold particle mixture is prepared. First, for 20 bombardments, 12 mg of gold is placed in a microfuge tube and 1 ml of 70% ethanol is added. Vortex at maximum speed for 5 minutes, then centrifuge for 5 seconds and remove the supernatant.

[0111] Add 1 ml of distilled water to the gold particles, vortex for 1 minute, let stand for 1 minute, centrifuge for 5 seconds, and then remove the supernatant. Repeat this sequence three times to wash the gold particles. Next, add 205 μl of 50% glycerol and vortex for 5 minutes to suspend the particles. While maintaining a vortex speed of 2-3, transfer 100 μl of gold particles to a new microfuge tube for 10 bombardments. Then, while vortexing, add 10 μl of DNA (0.5-20 μg / μl), 100 μl of 2.5 M CaCl , and 10 μl of 2.5 M CaCl . 2、Add 40 μl of 0.1M spermidine in this order and mix by pipetting. After mixing, continue vortexing for 2 minutes and let stand for 1 minute. Centrifuge for 2 seconds to sediment and remove the supernatant. After removing the supernatant, add 300 μl of 70% ethanol and wait 1 minute. After removing the supernatant, add 300 μl of 100% ethanol and wait 1 minute. Remove again, add 110 μl of 100% ethanol and vortex continuously to suspend the pellet.

[0112] Once the Gold Particle Mix is ​​complete, add 11 μl to the center of a microcarrier and let it dry for 5–10 minutes. Meanwhile, spread the prepared cells evenly over a 60 mm diameter medium. Next, place a stopping screen on the Brass Adjustable Nest and place the completely dried microcarriers on top. Then, secure it in place with the holder, turn on the gene gun, and preheat for 5 minutes. Once preheating is complete, attach the rupture disk to the retaining cap, screw it in, and insert it into the gun. Then, attach the Brass Adjustable Nest containing the microcarriers, place the medium containing the cells at Target Distance 3, and close the door. Start the helium gas supply and press the Vacuum button. When the vacuum reaches 29 inches Hg and the helium pressure reaches 1300 psi, press and hold the Fire button. When the pressure rises to 1100 psi, the gold particles will be fired. After bombardment, the cultures were incubated at 23°C for a recovery period.

[0113] Example 3: Confirmation of transformants of Chlorella vulgaris Biolistic transformation was performed in the same manner as in Example 2, and after subculture, it was confirmed that antibiotic resistance was maintained at various concentrations, as shown in Figure 7. A total of 42 transformants were obtained. These were subcultured using the medium used in Example 2 under light irradiation.

[0114] To confirm gene and protein expression in these transformants, whole protein SDS-PAGE was performed to confirm the band size of approximately 43 kDa of the target protein, beta-type carbonic anhydrase. The results are shown in Figure 8.

[0115] To confirm the band of this target protein, MALDI-TOF was performed, and the results are shown in FIG.

[0116] The present invention was advanced by confirming the expression of proteins in transformants and the maintenance of resistance through successive generations. For genetic confirmation, the presence or absence of the target gene was confirmed by Southern blot, and the results are shown in Figure 10.

[0117] As can be seen from FIG. 10, a DNA band was observed at the same position as the size of the target gene, and this was also confirmed by DNA sequencing by PCR.

[0118] Example 4: Sr biomineralization The functionality of the transformants was confirmed by microscopic visualization of biomineralized crystals in the same amount to provide improved Sr biomineralization. First, the Sr biomineralization ability of wild-type (WT) Chlorella vulgaris was confirmed.

[0119] Specifically, Chlorella vulgaris was cultured under light conditions until the OD reached 0.6. After the culture, the microalgae were harvested under sterilized conditions at 25°C, 4000 rpm, and 15 minutes. The microalgae were then washed three times with 3 mM NaHCO3. The cells were then collected at 1 x 10 7The solution was adjusted to 3 mM NaHCO3, and 200 ppm of Sr was added to the prepared solution. The prepared Chlorella vulgaris was then mixed and cultured at 4°C for at least 4 hours. The solution was then stained with 0.004% sodium rhodizonate and observed under a microscope. The results are shown in Figure 11.

[0120] As can be seen in Figure 11, crystallization of the WT resulted in a smaller amount of biomineralized crystals than that of the transformant. Furthermore, for quantitative comparison, analysis was performed by ICP-MS, and the results are shown in Figure 12 and Table 5.

[0121] [Table 5]

[0122] As can be seen from FIG. 12 and Table 5, the transformants were found to have biomineralization crystals that increased by up to about 160% compared to the wild type.

[0123] Example 5. Biomineralization Results of Lithium (Li) in Single LiCl

[0124] 5-1.Removal rate according to LiCl concentration The lithium crystal removal rate as a function of lithium concentration in a single LiCl solution was determined using the transformed Chlorella vulgaris cells from Example 3, LiCl (Sigma), sterilized MBM medium (KNO3 2.5 mM, MgSO4 7H2O 0.3 mM, K2HPO4 0.43 mM, KH2PO4 1.29 mM, NaCl 0.43 mM, CaCl2*2H2O 0.068 mM, FeSO4*7H2O 0.1 g, A5 metal mix ml / L), deionized water (DIW), white light, a stirrer, a 10 ml conical tube, a centrifuge, and a 0.2 μM syringe filter. The specific experimental method is as follows.

[0125] Fresh (log phase) cells of Chlorella vulgaris were placed in MBM medium at 6 x 10 ^ The cells were cultured at 7 / ml. Five ml of cells were collected from the medium and mixed with 5 ml of LiCl solution at different concentrations (300, 600, and 900 mM) in a 10 ml conical tube to make a total volume of 10 ml. The mixture was incubated under white light for 16 hours with gentle stirring. After 16 hours, the mixture was centrifuged at 3000 rpm for 3 minutes. The supernatant was collected and filtered to remove the cells and crystals using a 0.2 μM syringe filter. The collected solution was used for ICP analysis. The sample was loaded into an ICP-OES and MS instrument, where it was ionized using inductively coupled plasma (ICP) analysis. The ions were then separated using a mass analyzer and subjected to ICP analysis. The results are shown in Figures 13 and 14.

[0126] The lithium removal rate depending on the lithium concentration was confirmed from the results in Figure 13. It was confirmed that the lithium removal rate was approximately 71% at 300 mM, approximately 94% at 600 mM, and approximately 28% at 900 mM.

[0127] The lithium removal rate in the presence or absence of cells was also confirmed from the results in Figure 14. It was confirmed that the lithium removal rate in the presence of cells (w / C. vulgaris) was significantly higher than that in the absence of cells (w / o C. vulgaris) at all concentrations, and the specific measured values ​​in Figure 14 are shown in Table 6 below.

[0128] [Table 6]

[0129] 5-2.Effects on Sodium and Sulfate To measure the effects of sodium and sulfate, the same experiment as in Example 5-1 was performed, except that NaCl or potassium sulfate was added to the conical tube at different concentrations (300, 600, and 900 mM). The Li concentration was fixed at 600 mM. The results are shown in Figures 15 and 16.

[0130] As a result, the lithium removal rate was confirmed as a function of sodium concentration from the results in Figure 15. It was confirmed that the lithium removal rate was approximately 13% at 300 mM, approximately -3% at 600 mM, and approximately 17% at 900 mM. In other words, it was confirmed that there is no consistent correlation between sodium concentration and lithium removal rate.

[0131] The results in Figure 16 also confirmed the lithium removal rate depending on the sulfate concentration. It was confirmed that the lithium removal rate was approximately -12% at 300 mM, approximately -1% at 600 mM, and approximately 13% at 900 mM. In other words, it was confirmed that there was no consistent correlation between the sulfate concentration and the lithium removal rate.

[0132] Example 6. Preparation of Lithium Battery Solution Lithium battery solution was prepared by mixing LiCl (sigma), NiCl2 (sigma), CoCl2 (sigma), MnCl (sigma), AlCl3 (sigma), CuCl2 (sigma), FeCl2 (sigma), ClF (sigma), and KH2PO4 (sigma) with deionized water (DIW). The composition of the prepared lithium battery solution was analyzed and is shown in Table 7 below and Figure 17.

[0133] [Table 7]

[0134] As can be seen from Table 7, the Li content was determined to be 4% (1M), Ni 26% (1.09M), and Co 33% (1.39M).

[0135] Example 7. Biomineralization Results of Lithium (Li) in Lithium Battery Solution

[0136] 7-1. Crystallization in Lithium Battery Solutions The crystallization of Li in the lithium battery solution was confirmed using the Chlorella vulgaris cells transformed in Example 3, the lithium battery solution prepared in Example 6, sterilized MBM medium (2.5 mM KNO, 0.3 mM MgSO7H2O, 0.43 mM K2HPO4, 1.29 mM KH2PO4, 0.43 mM NaCl, 0.068 mM CaCl2*2H2O, 0.1 g FeSO4*7H2O, A5 metal mix ml / L), deionized water (DIW), white light, a stirrer, a 10 ml conical tube, a centrifuge, and a 0.2 μM syringe filter. The specific experimental method is as follows.

[0137] Fresh (log phase) cells of Chlorella vulgaris were cultured in MBM medium at a concentration of 6*10^7 / ml. Five ml of cells were harvested from the medium and mixed with 5 ml of 2 M LiCl in a 10 ml conical tube to prepare a final 1 M LiCl solution. The mixture was incubated under white light for 16 hours with gentle stirring. After 16 hours, the mixture was centrifuged at 3000 rpm for 3 minutes. The supernatant was collected and filtered to remove the cells and crystals using a 0.2 μM syringe filter. The collected solution was used for ICP analysis. The sample was loaded into an ICP-OES and MS instrument, where it was ionized using inductively coupled plasma (ICP), and the ions were separated using a mass analyzer for ICP analysis. The results are shown in Figure 18.

[0138] The lithium removal rate in the presence or absence of cells was confirmed from the results in Figure 18. When cells were not present (w / o Cell), almost no lithium was removed, but when cells were present (w / Cell), approximately 50% of the lithium was removed.

[0139] 7-2. Crystallization of Na and S in Lithium Battery Solutions To measure the effects of sodium and sulfate, the same experiment as in Example 7-1 was performed, except that NaCl or potassium sulfate was added to the conical tube at different concentrations (300, 600, 900 mM). The results are shown in Figures 19 and 20.

[0140] The results in Figure 19 confirmed that the lithium removal rate varied depending on the sodium concentration. At 300 mM, the lithium removal rate was approximately 56%, at 600 mM, approximately 68%, and at 900 mM, approximately 37%. In other words, it was confirmed that there was no consistent correlation between the sodium concentration and the lithium removal rate.

[0141] Furthermore, the results in Figure 20 confirmed that the lithium removal rate varied depending on the sulfate concentration. It was confirmed that the lithium removal rate was approximately 33% at 300 mM, approximately 45% at 600 mM, and approximately 75% at 900 mM. In other words, it was confirmed that the lithium removal rate increased as the sulfate concentration increased.

[0142] Example 8. Biomineralization results of Nickel (Ni) in single NiCl

[0143] 8-1.Removal rate according to NiCl2 concentration The nickel removal rate of a single NiCl solution was determined using the transformed Chlorella vulgaris cells from Example 3, NiCl (Sigma), sterile MBM medium (2.5 mM KNO, 0.3 mM MgSO 7H2O, 0.43 mM K2HPO4, 1.29 mM KH2PO4, 0.43 mM NaCl, 0.068 mM CaCl2*2H2O, 0.1 g FeSO4*7H2O, and A5 metal mix ml / L), deionized water (DIW), a white light, a stirrer, a 10 ml conical tube, a centrifuge, and a 0.2 μM syringe filter. The specific experimental method is as follows.

[0144] Fresh (log phase) cells of Chlorella vulgaris were cultured in MBM medium at a concentration of 6*10^7 / ml. Five ml of cells were harvested from the medium and mixed with 5 ml of NiCl2 solutions (300, 600, and 900 mM) in a 10 ml conical tube to make a total volume of 10 ml. The cells were incubated under white light for 16 hours with gentle stirring. After 16 hours, the cells were centrifuged at 3000 rpm for 3 minutes. The supernatant was collected and filtered to remove the cells and crystals using a 0.2 μM syringe filter. The collected solution was used for ICP analysis. The sample was loaded into an ICP-OES and MS instrument, where it was ionized by inductively coupled plasma (ICP), and the ions were separated using a mass analyzer for ICP analysis. The results are shown in Figures 21 and 22.

[0145] The nickel removal rate depending on the nickel concentration was confirmed from the results in Figure 21. It was confirmed that the nickel removal rate was approximately 36% at 300 mM, approximately 15% at 600 mM, and approximately 23% at 900 mM.

[0146] Furthermore, the nickel removal rate in the presence or absence of cells was confirmed from the results in Figure 22. It was confirmed that the nickel removal rate in the presence of cells (w / C. vulgaris) was significantly higher than that in the absence of cells (w / o C. vulgaris) at all concentrations. Specific measured values ​​in Figure 14 are shown in Table 8 below.

[0147] [Table 8]

[0148] 8-2.Effects on Sodium and Sulfate To measure the effects of sodium and sulfate, the same experiment as in Example 8-1 was performed, except that NaCl or potassium sulfate was added to the conical tube at different concentrations (300, 600, and 900 mM). The Ni concentration was fixed at 600 mM. The results are shown in Figures 23 and 24.

[0149] As a result, the nickel removal rate was confirmed as a function of sodium concentration from the results in Figure 23. It was confirmed that the nickel removal rate was approximately 11% at 300 mM, approximately 8% at 600 mM, and approximately 4% at 900 mM. In other words, it was confirmed that the nickel removal rate decreased as the sodium concentration increased.

[0150] Furthermore, the nickel removal rate was confirmed as a function of sulfate concentration from the results in Figure 24. It was confirmed that the nickel removal rate was approximately 6% at 300 mM, approximately 2% at 600 mM, and approximately 9% at 900 mM. In other words, it was confirmed that there was no consistent correlation between the sulfate concentration and the lithium removal rate.

[0151] Example 9. Preparation of Nickel Battery Solution A nickel battery solution was prepared by mixing LiCl2 (sigma), NiCl2 (sigma), CoCl2 (sigma), MnCl (sigma), AlCl3 (sigma), CuCl2 (sigma), FeCl2 (sigma), ClF (sigma), and KH2PO4 (sigma) with deionized water (DIW). The composition of the prepared nickel battery solution was analyzed and is shown in Table 9 below and Figure 25.

[0152] [Table 9]

[0153] As can be seen from Table 9, the Li content was determined to be 4% (1M), Ni content was determined to be 26% (1.09M), and Co content was determined to be 33% (1.39M).

[0154] Example 10. Biomineralization Results of Nickel (Ni) in Nickel Battery Solution

[0155] 10-1. Crystallization in Nickel Battery Solutions The results of Ni crystallization in the nickel battery solution were confirmed using the Chlorella vulgaris cells transformed in Example 3, the nickel battery solution prepared in Example 9, sterilized MBM medium (2.5 mM KNO, 0.3 mM MgSO 7H2O, 0.43 mM K2HPO4, 1.29 mM KH2PO4, 0.43 mM NaCl, 0.068 mM CaCl2*2H2O, 0.1 g FeSO4*7H2O, A5 ml / L metal mix), deionized water (DIW), a white light, a stirrer, a 10 ml conical tube, a centrifuge, and a 0.2 μM syringe filter. The specific experimental method is as follows.

[0156] Fresh (log phase) cells of Chlorella vulgaris were cultured in MBM medium at a concentration of 6*10^7 / ml. Five ml of cells were harvested from the medium and mixed with 5 ml of 2.18 M NiCl2 nickel battery solution in a 10 ml conical tube to prepare a final 10 ml solution of 1.09 M NiCl2. The mixture was incubated under white light for 16 hours with gentle stirring. After 16 hours, the mixture was centrifuged at 3000 rpm for 3 minutes. The supernatant was collected and filtered to remove the cells and crystals using a 0.2 μM syringe filter. The collected solution was used for ICP analysis. The sample was loaded into an ICP-OES and MS instrument, where it was ionized using inductively coupled plasma (ICP), and the ions were separated by a mass analyzer for ICP analysis. The results are shown in Figure 26.

[0157] The nickel removal rate in the presence or absence of cells was confirmed from the results in Figure 26. When cells were not present (w / o Cell), almost no nickel was removed, but when cells were present (w / Cell), approximately 80% of the nickel was removed.

[0158] 10-2. Crystallization of Na and S in Nickel Battery Solutions To measure the effects of sodium and sulfate, the same experiment as in Example 10-1 was performed, except that NaCl or potassium sulfate was added to the conical tube at different concentrations (300, 600, 900 mM). The results are shown in Figures 27 and 28.

[0159] The results in Figure 27 confirmed that the nickel removal rate varied depending on the sodium concentration. At 300 mM, the nickel removal rate was approximately 74%, at 600 mM, approximately 62%, and at 900 mM, approximately 80%. In other words, it was confirmed that there was no consistent correlation between the sodium concentration and the lithium removal rate.

[0160] Furthermore, the results in Figure 28 confirmed that the nickel removal rate varied depending on the sulfate concentration. At 300 mM, the nickel removal rate was approximately 12%, at 600 mM, approximately 55%, and at 900 mM, approximately 64%. In other words, it was confirmed that the lithium removal rate increased as the sulfate concentration increased.

[0161] Example 11. Biomineralization results of Cobalt (Co) in single CoCl

[0162] 11-1.Removal rate according to CoCl2 concentration The removal rate of cobalt from a single CoCl solution was determined using the transformed Chlorella vulgaris cells from Example 3, CoCl (Sigma), sterilized MBM medium (2.5 mM KNO, 0.3 mM MgSO 7H2O, 0.43 mM K2HPO4, 1.29 mM KH2PO4, 0.43 mM NaCl, 0.068 mM CaCl2*2H2O, 0.1 g FeSO4*7H2O, and A5 ml / L metal mix), deionized water (DIW), a white light, a stirrer, a 10 ml conical tube, a centrifuge, and a 0.2 μM syringe filter. The specific experimental method was as follows.

[0163] Fresh (log phase) cells of Chlorella vulgaris were cultured in MBM medium at a concentration of 6*10^7 / ml. Five ml of cells were harvested from the medium and mixed with 5 ml of CoCl2 solutions of different concentrations (300, 600, and 900 mM) in a 10 ml conical tube to make a total volume of 10 ml. The mixture was incubated under white light for 16 hours with gentle stirring. After 16 hours, the mixture was centrifuged at 3000 rpm for 3 minutes. The supernatant was collected and filtered to remove the cells and crystals using a 0.2 μM syringe filter. The collected solution was used for ICP analysis. The sample was loaded into an ICP-OES and MS instrument, where it was ionized by inductively coupled plasma (ICP), and the ions were separated using a mass analyzer for ICP analysis. The results are shown in Figures 29 and 30.

[0164] The cobalt removal rate depending on the cobalt concentration was confirmed from the results in Figure 29. It was confirmed that the cobalt removal rate was approximately 50% at 300 mM, approximately 54% at 600 mM, and approximately 30% at 900 mM.

[0165] Furthermore, the results in Figure 30 confirmed the cobalt removal rate depending on whether or not cells were present. It was confirmed that at all concentrations, the cobalt removal rate was significantly higher when cells were present (w / C. vulgaris) than when cells were not present (w / o C. vulgaris). Specific measured values ​​in Figure 14 are shown in Table 10 below.

[0166] [Table 10]

[0167] 11-2.Effects on Sodium and Sulfate To measure the effects of sodium and sulfate, the same experiment as in Example 11-1 was performed, except that NaCl or potassium sulfate was added to the conical tube at different concentrations (300, 600, and 900 mM). The Co concentration was fixed at 600 mM. The results are shown in Figures 31 and 32.

[0168] As a result, the cobalt removal rate was confirmed as a function of sodium concentration from the results in Figure 31. It was confirmed that the cobalt removal rate was approximately 3% at 300 mM, approximately 0% at 600 mM, and approximately 3% at 900 mM. In other words, it was confirmed that there was no consistent correlation between sodium concentration and cobalt removal rate.

[0169] Furthermore, the results in Figure 32 confirmed that the cobalt removal rate varied depending on the sulfate concentration. It was confirmed that the cobalt removal rate was approximately 5% at 300 mM, approximately 13% at 600 mM, and approximately 4% at 900 mM. In other words, it was confirmed that there was no consistent correlation between the sulfate concentration and the cobalt removal rate.

[0170] Example 12. Preparation of Cobalt Battery Solution A cobalt battery solution was prepared by mixing LiCl2 (sigma), NiCl2 (sigma), CoCl2 (sigma), MnCl(sigma), AlCl3 (sigma), CuCl2 (sigma), FeCl2 (sigma), ClF (sigma), and KH2PO4 (sigma) with deionized water (DIW). The composition of the prepared cobalt battery solution was analyzed and is shown in Table 11 below and Figure 33.

[0171] [Table 11]

[0172] As can be seen from Table 11, the Li was found to be 4% (1M), Ni 26% (1.09M), and Co 33% (1.39M).

[0173] Example 13. Biomineralization Results of Cobalt (Co) in Cobalt Battery Solution

[0174] 13-1. Crystallization in cobalt battery solutions The results of Co crystallization in the cobalt battery solution were confirmed using the Chlorella vulgaris cells transformed in Example 3, the cobalt battery solution prepared in Example 12, sterilized MBM medium (2.5 mM KNO, 0.3 mM MgSO 7H2O, 0.43 mM K2HPO4, 1.29 mM KH2PO4, 0.43 mM NaCl, 0.068 mM CaCl2*2H2O, 0.1 g FeSO4*7H2O, A5 ml / L metal mix), deionized water (DIW), a white light, a stirrer, a 10 ml conical tube, a centrifuge, and a 0.2 μM syringe filter. The specific experimental method is as follows.

[0175] Fresh (log phase) cells of Chlorella vulgaris were cultured in MBM medium at a concentration of 6*10^7 / ml. Five ml of cells were harvested from the medium and mixed with 5 ml of 2.78 M NiCl2 cobalt battery solution in a 10 ml conical tube to prepare a final 10 ml solution of 1.39 M CoCl2. The mixture was incubated under white light for 16 hours with gentle stirring. After 16 hours, the mixture was centrifuged at 3000 rpm for 3 minutes. The supernatant was collected and filtered to remove the cells and crystals using a 0.2 μM syringe filter. The collected solution was used for ICP analysis. The sample was loaded into an ICP-OES and MS instrument, where it was ionized using inductively coupled plasma (ICP) analysis. The ions were then separated by a mass analyzer and analyzed using ICP analysis. The results are shown in Figure 34.

[0176] The cobalt removal rate in the presence or absence of cells was confirmed from the results in Figure 34. When cells were not present (w / o Cell), almost no cobalt was removed, but when cells were present (w / Cell), approximately 75% of the cobalt was removed.

[0177] 13-2. Crystallization of Cobalt Battery Solutions with Na and S To measure the effects of sodium and sulfate, the same experiment as in Example 13-1 was performed, except that NaCl or potassium sulfate was added to the conical tube at different concentrations (300, 600, 900 mM). The results are shown in Figures 35 and 36.

[0178] The results in Figure 35 confirmed that the cobalt removal rate varied depending on the sodium concentration. At 300 mM, the cobalt removal rate was approximately 74%, at 600 mM, approximately 64%, and at 900 mM, approximately 77%. In other words, it was confirmed that there was no consistent correlation between the sodium concentration and the cobalt removal rate.

[0179] Furthermore, the results in Figure 36 confirmed that the cobalt removal rate varied depending on the sulfate concentration. It was confirmed that the cobalt removal rate was approximately 13% at 300 mM, approximately 53% at 600 mM, and approximately 62% at 900 mM. In other words, it was confirmed that the cobalt removal rate increased as the sulfate concentration increased.

[0180] small knot The biomineralization results for lithium (Li) in simple LiCl solution and lithium battery solution are summarized and shown in Tables 12 and 13 below.

[0181] [Table 12]

[0182] [Table 13]

[0183] The biomineralization results for Nickel (Ni) in a single NiCl2 solution and in nickel battery solution were summarized and shown in Tables 14 and 15 below.

[0184] [Table 14]

[0185] [Table 15]

[0186] The biomineralization results of Cobalt (Co) in a single CoCl2 solution and a cobalt battery solution were summarized and shown in Tables 16 and 17 below.

[0187] [Table 16]

[0188] [Table 17] [Industrial Applicability]

[0189] The present invention relates to a composition for leaching lithium, nickel, or cobalt using a Chlorella vulgaris strain, and a method for leaching lithium, nickel, or cobalt.

Claims

1. A composition for leaching lithium, nickel, or cobalt, comprising at least one member selected from the group consisting of a Chlorella vulgaris strain, a culture of said strain, a concentrate of said culture, and a dried product of said culture.

2. 2. The lithium, nickel or cobalt leaching composition of claim 1, wherein the strain is transformed with a vector comprising: Coccomyxa C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo) small subunit 2 (rbcS2) gene promoter; Coccomyxa subellipsoidea C-169 beta-type carbonic anhydrase-encoding nucleotide sequence; and Terminator sequence of the rbcS2 gene of Coccomyxa C-169.

3. 3. The lithium, nickel or cobalt leaching composition of claim 2, wherein the promoter comprises the base sequence of SEQ ID NO:

2.

4. The lithium, nickel or cobalt leaching composition according to claim 2, wherein the beta-type carbonic anhydrase-encoding nucleotide sequence of Coccomyxa subellipsoidea C-169 is operatively linked to a promoter.

5. 3. The lithium, nickel or cobalt leaching composition of claim 2, wherein the terminator sequence comprises the base sequence of SEQ ID NO:

3.

6. 3. The lithium, nickel or cobalt leaching composition of claim 2, wherein the vector further comprises an antibiotic resistance gene as a selectable marker.

7. 7. The lithium, nickel or cobalt leaching composition according to claim 6, wherein the antibiotic is one or more selected from the group consisting of spectinomycin, paromomycin, ampicillin, zeocin and bleomycin.

8. The lithium, nickel or cobalt leaching composition of claim 2 , wherein the vector further comprises a gene encoding a reporter molecule.

9. 9. The lithium, nickel or cobalt leaching composition according to claim 8, wherein the reporter molecule is one or more selected from the group consisting of growth-promoting proteins, fluorescent proteins and hydrolases.

10. 10. The lithium, nickel or cobalt leaching composition of claim 9, wherein the fluorescent protein is luciferase.

11. 10. The lithium, nickel or cobalt leaching composition of claim 9, wherein the hydrolytic enzyme is β-glucuronidase.

12. 3. The composition for leaching lithium, nickel or cobalt according to claim 2, wherein the vector is for transformation of microalgae using gold particles bombardment.

13. a mixing step of mixing one or more selected from the group consisting of a Chlorella vulgaris strain, a culture of the strain, a concentrate of the culture, and a dried product of the culture with a lithium-containing substance; and A method for leaching lithium, nickel or cobalt by bioleaching, comprising a leaching step for leaching lithium for a predetermined period of time.

14. 14. The lithium, nickel or cobalt leaching method according to claim 13, further comprising a transformation step prior to the mixing step, wherein the Chlorella vulgaris strain in the mixing step is a transformed Chlorella vulgaris strain: A transformation step of introducing a vector containing the promoter of the Coccomyxa C-169 ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo) small subunit 2 (rbcS2) gene, the beta-type carbonic anhydrase-encoding nucleotide sequence of Coccomyxa subellipsoidea C-169, and the terminator sequence of the Coccomyxa C-169 rbcS2 gene into Chlorella vulgaris.

15. 15. The lithium, nickel or cobalt leaching method of claim 14, wherein the promoter comprises the base sequence of SEQ ID NO:

2.

16. 15. The method for leaching lithium, nickel or cobalt according to claim 14, wherein the Coccomyxa subellipsoidea C-169 beta-type carbonic anhydrase-encoding nucleotide sequence is operatively linked to a promoter.

17. 15. The lithium, nickel or cobalt leaching method of claim 14, wherein the terminator sequence comprises the base sequence of SEQ ID NO:

3.

18. 15. The lithium, nickel or cobalt leaching method of claim 14, wherein the vector further comprises an antibiotic resistance gene as a selectable marker.

19. 19. The method of claim 18, wherein the antibiotic is one or more selected from the group consisting of spectinomycin, paromomycin, ampicillin, zeocin, and bleomycin.

20. 15. The method of claim 14, wherein the vector further comprises a gene encoding a reporter molecule.

21. 21. The method of claim 20, wherein the reporter molecule is one or more selected from the group consisting of growth-promoting proteins, fluorescent proteins, and hydrolytic enzymes.

22. 22. The lithium, nickel or cobalt leaching method of claim 21, wherein the fluorescent protein is luciferase.

23. 22. The lithium, nickel or cobalt leaching method of claim 21, wherein the hydrolytic enzyme is β-glucuronidase.

24. 15. The method of claim 14, wherein the vector is for transformation of microalgae using gold particles bombardment.

25. 15. The method of claim 14, wherein the transformation step is carried out using gold particles bombardment.

26. 15. A lithium, nickel or cobalt leaching process according to claim 14, wherein the cell stage of the transformation step is the log phase.

27. 27. A lithium, nickel or cobalt leaching process according to claim 26, wherein the log phase has an OD686 value of 0.4 to 0.

6.

28. The cell density in the transformation step was 5.0*10 ６ ~8.0*10 ７ 15. The lithium, nickel or cobalt leaching method of claim 14, wherein the diameter of the leaching agent is per 60 mm.

29. 15. The lithium, nickel or cobalt leaching process of claim 14, wherein the vacuum in the transformation step is 27.0 to 29.0 inches Hg.

Images