A new proteus vulgaris strain capable of efficiently recovering rare earth ions from leaching solution and mineralizing rare earth phosphate in cells

Abstract

The present application relates to the field of environment-friendly biological rare earth recovery technology, in particular to Methylobacterium extorquens C2 and a method for leaching rare earth elements from waste materials, in particular, the strain provided by the present application uses microorganisms and their metabolites to leach rare earth elements from waste materials and mineralize nanometer or micrometer rare earth phosphate in cells, solving the problems of large amount of chemical reagent, high production cost and serious environmental pollution in the process of chemical leaching of rare earth waste materials. The strain provided by the present application has low cultivation cost, fast growth speed and simple leaching conditions, and has leaching capacity for rare earth ions in various waste materials, and has good application prospect in the aspects of microbial recovery and reuse of rare earth waste materials.

Description

Technical Field

[0001] This invention relates to the field of environmentally friendly biological rare earth recycling technology, and in particular to a novel *Tricholoma materia elegans* strain that can efficiently recover rare earth ions from waste leachate and mineralize nano- or micron-sized rare earth phosphates intracellularly, as well as its leaching method. Background Technology

[0002] Rare earth elements, including fifteen lanthanides and scandium and yttrium, are widely used as important strategic resources in cutting-edge research and high-tech industries, and are known as "industrial gold." However, due to their uneven distribution and limited reserves, with the widespread application of rare earth elements in permanent magnet materials, industrial catalysis, and other fields, it has become essential to extract rare earth elements from these secondary rare earth resources.

[0003] Traditional methods for recovering and enriching rare earth elements from urban mines primarily employ physicochemical approaches using strong acids and organic solvents. However, these methods are complex, energy-intensive, and cause significant environmental pollution. Therefore, there is an urgent need to develop a clean and efficient new technology for rare earth enrichment to ensure both the enrichment rate and purity of rare earth elements while minimizing environmental pollution.

[0004] Biospecific intracellular mineralization enrichment using methyltrophic microorganisms is an environmentally friendly alternative. Methanol dehydrogenase (MDH) is considered a key and essential enzyme in methanol metabolism in *Methylorubrum extorquens* strains. Recent studies have shown that *M. extorquens* contains XoxF-type MDH with lanthanides as cofactors, and a series of proteins and organic molecules that ensure the correct binding and storage of lanthanides. This system can be rationally designed and modified for the recovery and separation of rare earth ions based on the *M. extorquens* system. However, commercial methyltrophic microorganisms such as *M. extorquens AM1*, used as model strains, have low efficiency and are time-consuming in recovering rare earth elements from leachates of spent FCC catalysts and NdFeB magnets. This is mainly because these microorganisms have poor tolerance to high concentrations of rare earth ions. Therefore, screening and culturing robust microorganisms from methyltrophic microbial communities associated with rare earth mines and rare earth waste is crucial for improving the bioaccumulation efficiency of rare earth elements. However, reports on bioleaching based on such microorganisms are limited. Therefore, providing a novel *Tricholoma mater* strain that can efficiently recover rare earth ions from waste leachate and mineralize nano- or micro-sized rare earth phosphates intracellularly can offer new options and pathways for biometallurgy and has significant practical implications. Summary of the Invention

[0005] In view of this, the present invention provides a novel *Tricholoma mater* strain that can efficiently recover rare earth ions from waste leachate and mineralize nano- or micron-sized rare earth phosphates intracellularly, thus solving the problems of large chemical reagent consumption, high production costs, and serious environmental pollution in the chemical recycling process of rare earth waste.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides Methylobacterium extorquens C2, which has the accession number CGMCC No. 30396.

[0008] Secondly, the present invention also provides a method for culturing the aforementioned Methylobacterium extorquens C2, wherein the Methylobacterium extorquens C2 is picked and cultured in liquid culture medium supplemented with tryptic soy broth (TSB) at 30°C and 200 rpm for 2 to 4 days.

[0009] Thirdly, the present invention also provides one or more of the inactivated bacterial cells, exosomes, or metabolites of Methylobacterium extorquens C2.

[0010] Fourthly, the present invention also provides a method for preparing the microbial culture medium of Methylobacterium extorquens C2, wherein the Methylobacterium extorquens C2 is picked and cultured in liquid culture medium supplemented with tryptic soy broth (TSB) at 30°C and 200 rpm for 2 to 4 days to obtain the microbial culture medium.

[0011] Fifthly, the present invention also provides a microbial culture medium prepared by the aforementioned preparation method.

[0012] In a sixth aspect, the present invention also provides the use of any of the following in leaching wastes of rare earth elements and / or in intracellular mineralization of nano or micron-sized rare earth phosphates;

[0013] (I) Methylobacterium extorquens C2;

[0014] (II) One or more of the inactivated bacterial cells, exosomes or metabolites mentioned above;

[0015] (III) The microbial culture medium.

[0016] In some specific embodiments of the present invention, the waste includes one or more of waste FCC catalyst, waste NdFeB sludge, or waste NdFeB powder.

[0017] In some specific embodiments of the present invention, the main framework of the spent FCC catalyst is aluminosilicate, containing rare earth elements and / or transition elements, wherein the content of the rare earth elements and / or transition elements is 0.01% to 10%; the rare earth elements include, but are not limited to, lanthanum and / or cerium; the transition elements include, but are not limited to, nickel and / or vanadium.

[0018] The main component of the waste NdFeB sludge is NdFeB (N2Fe). 14 B) Magnets containing 1% to 30% lubricating oil;

[0019] The main component of the waste NdFeB powder is NdFeB (Nd2Fe). 14 B) Originates from various stages of industrial production of NdFeB magnets, including cutting, molding, or sintering processes.

[0020] In a seventh aspect, the present invention also provides an article of manufacture comprising any one of the following:

[0021] (I) Methylobacterium extorquens C2;

[0022] (II) One or more of the inactivated bacterial cells, exosomes or metabolites mentioned above;

[0023] (III) The aforementioned microbial culture medium;

[0024] The products include microecological products, probiotic products, synbiotic products and / or postbiotic products.

[0025] Eighthly, the present invention also provides a method for leaching rare earth elements from leaching waste and / or mineralizing nano- or micron-sized rare earth phosphates intracellularly, wherein any one of the following is mixed with the waste and incubated;

[0026] (I) Methylobacterium extorquens C2;

[0027] (II) One or more of the inactivated bacterial cells, exosomes or metabolites mentioned above;

[0028] (III) The aforementioned microbial culture medium;

[0029] (IV) The aforementioned product.

[0030] In some specific embodiments of the present invention, the waste includes one or more of waste FCC catalyst, waste NdFeB sludge, or waste NdFeB powder;

[0031] Preferably, the main framework of the spent FCC catalyst is aluminosilicate, containing rare earth elements and / or transition elements, wherein the content of the rare earth elements and / or transition elements is 0.01% to 10%; the rare earth elements include, but are not limited to, lanthanum and / or cerium; the transition elements include, but are not limited to, nickel and / or vanadium.

[0032] The main component of the waste NdFeB sludge is NdFeB (N2Fe). 14 B) Magnets containing 1% to 30% lubricating oil;

[0033] The main component of the waste NdFeB powder is NdFeB (Nd2Fe). 14 B) Originates from various stages of industrial production of NdFeB magnets, including cutting, molding, or sintering processes.

[0034] Preferably, the liquid-to-solid ratio of the Methylobacterium extorquens C2 to the spent FCC catalyst is 1:(1-100);

[0035] The liquid-to-solid ratio of the Methylobacterium extorquens C2 to the waste NdFeB sludge is 1:(1-100);

[0036] The liquid-to-solid ratio of the Methylobacterium extorquens C2 to the waste NdFeB powder is 1:(1-100);

[0037] Preferably, the incubation conditions are as follows: cultured in tryptic soy broth (TSB) liquid medium at 30°C and 200 rpm for 2–4 days, and the cells are collected by centrifugation at 8000 rpm for 5 min, and then washed with water to obtain rare earth enriched cells.

[0038] This invention provides a novel *Tricholoma materata* strain capable of efficiently recovering rare earth ions from waste leachates and mineralizing nano- or micron-sized rare earth phosphates intracellularly. This addresses the problems of large quantities of chemical reagents, high production costs, and severe environmental pollution associated with the chemical recycling of rare earth waste. The strain provided by this invention has low cultivation costs, rapid growth rates, and simple enrichment conditions. Furthermore, it exhibits selective enrichment capabilities for rare earth ions in various waste leachates, demonstrating promising application prospects in the microbial recovery and reuse of rare earth waste.

[0039] Biological Preservation Instructions

[0040] Strain: C2; deposit date: April 22, 2024; deposit number: CGMCC No. 30397; classification name: Methylobacterium extorquens; depositary institution: China General Microbiological Culture Collection Center; depositary address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0042] Figure 1 The ICP-OES results show the enrichment of rare earth elements in the leachate of spent FCC catalyst by strain C2. The enrichment efficiency for La was 99.02%, and the enrichment efficiency for Ce was 96.82%.

[0043] Figure 2 The ICP results show the enrichment of rare earth elements in the leachate of waste NdFeB sludge by strain C2. The enrichment efficiency for Nd was 98.60%, for Gd it was 98.55%, for Tb it was 98.94%, and for Dy it was 98.95%.

[0044] Figure 3 The ICP results show the enrichment of rare earth elements in the leachate of waste NdFeB powder by strain C2. The enrichment efficiency for Nd was 99.56%, for Gd it was 98.45%, for Tb it was 98.37%, and for Dy it was 98.08%.

[0045] Figure 4 HRTEM image showing the mineralization of rare earth elements in the leachate of spent FCC catalyst by strain C2;

[0046] Figure 5 HRTEM image showing the mineralization of rare earth elements in waste NdFeB sludge leachate by strain C2. Detailed Implementation

[0047] This invention discloses a novel *Tricholoma materata* strain capable of efficiently recovering rare earth ions from waste leachate and mineralizing nano- or micron-sized rare earth phosphates intracellularly, along with its leaching method. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the same result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0048] The present invention adopts the following technical solution:

[0049] (1) Screening of methylotrophic strains: Strains were screened using MM medium solid plates containing methanol (0.1-5% v / v) and rare earth elements (1 μM-50 mM). Methylorubrum extorquens C2 (CGMCC No. 30397), which can grow on MM medium solid plates containing methanol and rare earth elements, was selected for further study.

[0050] (2) Microbial culture: Thymosin C2 was cultured in liquid culture medium supplemented with tryptic soy broth (TSB) at 30°C and 200 rpm for 2-4 days to obtain microbial culture medium.

[0051] (3) Microbial enrichment of rare earth elements in the leachate of spent FCC catalyst (the main framework is aluminosilicate, containing rare earth elements such as lanthanum and cerium, as well as transition elements such as nickel and vanadium, with a content of 0.01%-10%) (the source is a solution containing rare earth elements to be recovered generated during the microbial leaching of spent FCC catalyst, the same below): The culture medium of *Tymplocos nigra* C2 obtained in (2) was co-cultured with the leachate of spent FCC catalyst at a ratio of 1:1-1:100 in tryptic soy broth (TSB) liquid medium for 2-4 days at 30℃ and 200 rpm. The cells were collected by centrifugation at 8000 rpm for 5 min, and the cells were washed with water 3 times to obtain rare earth enriched cells.

[0052] (4) Microbial enrichment of waste NdFeB sludge (the main component of which is NdFeB (N2Fe) 14B) Rare earth elements in the leachate (from the process of microbial leaching of waste NdFeB sludge, containing 1%-30% lubricating oil, and containing rare earth elements to be recovered, hereinafter the same): The culture medium of *Tricholoma mater* C2 obtained in (2) was co-cultured with the leachate of waste NdFeB sludge at a ratio of 1:1-1:100 using tryptic soy broth (TSB) liquid medium at 30℃ and 200 rpm for 2-4 days. The cells were collected by centrifugation at 8000 rpm for 5 min, and the cells were washed 3 times with water to obtain rare earth enriched cells.

[0053] (5) Microbial enrichment of waste NdFeB powder (the main component of which is NdFeB (Nd2Fe) 14 B), rare earth elements generated in various stages of industrial NdFeB magnet production, such as cutting, molding, sintering, etc.) leachate (the source is a solution containing rare earth elements to be recovered generated during the microbial leaching of waste NdFeB powder, the same below): The culture medium of *Typhonium methylobacterium* C2 obtained in (2) was co-cultured with the waste NdFeB powder leachate at a ratio of 1:1 to 1:100 in tryptic soy broth (TSB) liquid medium for 2-4 days at 30℃ and 200rpm. The cells were collected by centrifugation at 8000rpm for 5min, and the cells were washed with water 3 times to obtain rare earth enriched cells.

[0054] (6) The enrichment and purification ability of C2 was identified by detecting the content and types of rare earth elements in the enriched bacterial cells in steps (3), (4) and (5) using ICP-OES and ICP-MS.

[0055] (7) The intracellular mineralization of rare earth phosphate in the enriched bacterial cells in steps (3), (4) and (5) is detected by TEM and HRTEM, and the mineralization type and effect are determined by comparing the lattice with the standard card.

[0056] The strain provided by this invention has low culture cost, fast growth rate, and simple enrichment conditions. It also has selective enrichment ability for rare earth ions in leachates of various wastes, and has good application prospects in the microbial recycling and reuse of rare earth waste.

[0057] The present invention provides a novel *Tricholoma materata* strain that can efficiently recover rare earth ions from waste leachate and mineralize nano- or micron-sized rare earth phosphates intracellularly, and the raw materials and reagents used in the leaching method are all commercially available.

[0058] The present invention will be further illustrated below with reference to the embodiments:

[0059] Example 1: Screening of Methyltrophic Strains

[0060] A single colony of purified *Methylobacterium extorquens* C2 was picked from MM solid medium containing 0.5% (v / v) methanol and 100 μM rare earth elements and added to 4 mL of MM liquid medium containing 0.5% (v / v) methanol and 100 μM rare earth elements. The culture was incubated overnight at 30°C and 200 rpm in a constant temperature shaking incubator. 2 μL of the bacterial culture was then added dropwise to an MM solid medium plate containing 0.5% (v / v) methanol and 100 μM rare earth elements. After five days of incubation, the results were observed and recorded. The presence of a distinct colony was recorded as positive, indicating that the strain possessed the ability for methyl vegetative growth. The culture was then biologically preserved, with the preservation number CGMCC No. 30397, and classified as *Methylobacterium extorquens*.

[0061] Example 2: Microbial co-culture enrichment of rare earth elements in waste FCC catalyst leachate

[0062] A single colony of purified C2 (accession number CGMCC No. 30397) was picked from MM solid medium containing 0.5% (v / v) methanol and 100 μM rare earth elements and added to 4 mL of TSB liquid medium. The culture was incubated overnight at 30°C and 200 rpm in a shaker to obtain the seed culture. 1% (v / v) of the seed culture was inoculated into TSB liquid medium containing a leachate of spent FCC catalyst (main framework of aluminosilicate, containing rare earth elements such as lanthanum and cerium, and transition elements such as nickel and vanadium, at a ratio of 1:1 to 1:100, with a content of 0.01% to 10%). The culture was incubated at 30°C and 200 rpm for 2-4 days. The cells were collected by centrifugation at 8000 rpm for 5 min, washed three times with water, and then the rare earth-enriched cells were obtained.

[0063] Example 3C2 Microbial co-culture enrichment of rare earth elements in leachate from waste NdFeB sludge

[0064] A single colony of purified C2 (CGMCC No. 30397) was picked from MM solid medium containing 0.5% (v / v) methanol and 100 μM rare earth elements and added to 4 mL of TSB liquid medium. The culture was incubated overnight at 30°C and 200 rpm in a shaker to prepare the seed culture. A 1% (v / v) portion of the seed culture was then inoculated into TSB liquid medium containing waste NdFeB sludge (mainly composed of NdFeB) at a ratio of 1:1 to 1:100. 14 B) Magnet extract containing 1%-30% lubricating oil. Incubate at 30℃ and 200rpm in a constant temperature shaking incubator for 2-4 days. Collect bacterial cells by centrifugation at 8000rpm for 5 minutes, and wash three times with water to obtain rare earth-enriched bacterial cells.

[0065] Example 4: Microbial co-culture enrichment of rare earth elements in leachate of waste NdFeB powder.

[0066] A single colony of purified C2 (accession number CGMCC No. 30397) was picked from MM solid medium containing 0.5% (v / v) methanol and 100 μM rare earth elements and added to 4 mL of TSB liquid medium. The culture was incubated overnight at 30°C and 200 rpm in a constant temperature shaking incubator to prepare the seed culture. A 1% (v / v) portion of the seed culture was then inoculated into TSB liquid medium containing waste NdFeB powder (mainly composed of NdFeB) at a ratio of 1:1 to 1:100. 14 B) Leachate produced in various stages of industrial NdFeB magnet production, such as cutting, molding, and sintering processes. The leachate was cultured at 30℃ and 200rpm in a constant-temperature shaking incubator for 2-4 days. The cells were collected by centrifugation at 8000rpm for 5 minutes, washed three times with water, and then the rare-earth-enriched cells were obtained.

[0067] Example 5: Detection of C2 enrichment ability by ICP-OES and ICP-MS

[0068] The enriched bacterial cells from Examples 2, 3, and 4 were digested using a nitric acid-perchloric acid (10:1) mixed acid system on a 210°C hot plate. After the solution became clear and transparent, it was diluted to a final volume to achieve the target element concentration of 1 ppb-10 ppm. ICP-OES and ICP-MS were used for qualitative and quantitative analysis of rare earth elements and other metal elements in the digestion solution. The experimental results are shown in [Figure number missing]. Figure 1 , 2, 3.

[0069] Table 1. ICP-OES results of rare earth element enrichment in waste FCC catalyst leachate by strain C2.

[0070]

[0071] Table 2 shows the ICP-OES results of rare earth element enrichment in waste NdFeB sludge leachate by strain C2.

[0072]

[0073] Table 3 shows the ICP-OES results of rare earth element enrichment in waste NdFeB powder leachate by strain C2.

[0074]

[0075] Example 6: Detection of C2-enriched phosphate crystals by HRTEM

[0076] The intracellular mineralization of rare earth phosphate at the nano or micron level in the enriched bacterial cells of Examples 3, 4, and 5 was detected by HRTEM. The elemental composition of the minerals was determined by EDS, and the mineralization type and effect were determined by lattice comparison standard cards. Quantitative analysis of the composition and purity was performed. The experimental results are shown in [Figure number missing]. Figure 4 5. Microorganisms mineralized rare earth element phosphate crystals within their cells, with interplanar spacings of 0.2247 nm and 0.3324 nm, corresponding to the interplanar spacings of lanthanum phosphate (031) and neodymium phosphate (002), respectively. EDS elemental analysis determined the composition of the rare earth phosphate crystals.

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Methylobacterium extorquens C2, characterized in that, Its accession number is CGMCC No.30397.

2. The method for culturing Methylobacterium extorquens C2 as described in claim 1, characterized in that, Select the Methylobacterium extorquens C2 and culture it in liquid culture medium supplemented with tryptic soy broth (TSB) at 30 ºC and 200 rpm for 2-4 days.

3. The method for preparing the microbial culture medium of Methylobacterium extorquens C2 as described in claim 1, characterized in that, Select the Methylobacterium extorquens C2 and culture it in liquid culture medium supplemented with tryptic soy broth (TSB) at 30 ºC and 200 rpm for 2-4 days to obtain a microbial culture medium.

4. The microbial culture medium prepared by the method described in claim 3.

5. The application of any of the following in the leaching waste of rare earth elements and / or in the intracellular mineralization of nano or micron-sized rare earth phosphates; (I) Methylobacterium extorquens C2 as described in claim 1; (II) The microbial culture medium as described in claim 4; The intracellular mineralization of nano- or micro-sized rare earth phosphates involves the intracellular mineralization of lanthanum phosphate and neodymium phosphate. The waste material is selected from one or more of the following: waste FCC catalyst, waste NdFeB sludge, or waste NdFeB powder; The main framework of the spent FCC catalyst is aluminosilicate, containing rare earth elements and / or transition elements, wherein the content of the rare earth elements and / or transition elements is 0.01%~10%; the rare earth elements are lanthanum and / or cerium; and the transition elements are nickel and / or vanadium. The main component of the waste NdFeB sludge is NdFeB (N2Fe). 14 B) Magnets containing 1% to 30% lubricating oil; The main component of the waste NdFeB powder is NdFeB (Nd2Fe). 14 B), which arises from various stages of industrial production of NdFeB magnets, including cutting, molding, or sintering processes.

6. A method for leaching rare earth elements and / or mineralizing nano- or micron-sized rare earth phosphates intracellularly from waste, characterized in that, Mix any of the following with the waste and incubate; (I) Methylobacterium extorquens C2 as described in claim 1; (II) The microbial culture medium as described in claim 4; The intracellular mineralization of nano- or micro-sized rare earth phosphates involves the intracellular mineralization of lanthanum phosphate and neodymium phosphate. The waste material is selected from one or more of the following: waste FCC catalyst, waste NdFeB sludge, or waste NdFeB powder; The main framework of the spent FCC catalyst is aluminosilicate, containing rare earth elements and / or transition elements, wherein the content of the rare earth elements and / or transition elements is 0.01%~10%; the rare earth elements are lanthanum and / or cerium; and the transition elements are nickel and / or vanadium. The main component of the waste NdFeB sludge is NdFeB (N2Fe). 14 B) Magnets containing 1% to 30% lubricating oil; The main component of the waste NdFeB powder is NdFeB (Nd2Fe). 14 B), which arises from various stages of industrial production of NdFeB magnets, including cutting, molding, or sintering processes.

7. The method as described in claim 6, characterized in that, The liquid-to-solid ratio of the Methylobacterium extorquens C2 to the spent FCC catalyst is 1:(1~100). The liquid-to-solid ratio of the Methylobacterium extorquens C2 to the waste NdFeB sludge is 1:(1~100). The liquid-to-solid ratio of the Methylobacterium extorquens C2 to the waste NdFeB powder is 1:(1~100).

8. The method as described in claim 6, characterized in that, The incubation conditions were as follows: cultured in tryptic soy broth (TSB) liquid medium at 30 ºC and 200 rpm for 2-4 days, and the cells were collected by centrifugation at 8000 rpm for 5 min. After washing with water, rare earth enriched cells were obtained.

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