Method for artificially constructing rare earth element leaching function synthetic flora

Abstract

The invention relates to a method for artificially constructing a rare earth element leaching function synthetic flora. The method comprises the following steps: enriching and domesticating a rare earth element leaching flora, determining a rare earth element leaching key functional flora, constructing a rare earth element leaching functional strain resource library, screening and determining an initial member of a leaching functional synthetic flora, and optimizing the configuration of the leaching functional synthetic flora to finally obtain the optimal leaching functional synthetic flora. According to the method provided by the invention, the key mineral leaching function microorganisms are searched based on a high-throughput sequencing technology, the interaction among the strains is fully exerted, and the adaptive capacity and stability of the strains to the stress environment of a mineral leaching system are improved, so that the leaching effect of improving the leaching rate of rare earth metal elements is realized. Moreover, a high-throughput sequencing technology and a pure culture technology are utilized, and concepts of synthetic biology and ecology are combined, so that a new way is provided for green and sustainable development and utilization of ionic rare earth elements, and a reference is also provided for construction of functional florae leached from other metal elements.

Description

Technical Field

[0001] The invention belongs to the technical field of biometallurgy and relates to a method for artificially constructing a synthetic bacterial community with rare earth element leaching function. Background Art

[0002] Rare earth elements are relatively abundant in the earth's crust, including 15 lanthanide elements and scandium (Sc) and yttrium (Y) with similar physical and chemical properties to lanthanide elements, totaling 17. Due to their unique physical and chemical properties, such as magnetism, fluorescence and superconductivity, rare earth elements play an important role in high-tech fields such as new energy, aerospace and military, and are regarded as important non-renewable strategic resources.

[0003] Industrial leaching of ionic rare earths usually leads to a series of ecological and environmental problems in mining areas, such as soil structure destruction, soil acidification and nutrient loss. In order to reduce the damage to the ecological environment caused by current methods, it is urgent to develop safe, pollution-free, green and efficient leaching technology.

[0004] Microbial leaching technology is particularly suitable for the development of rare earth resources in low-grade ores due to its advantages such as being green and low-cost. However, currently, single strains or simply superimposed mixed bacterial communities are mainly used to leach rare earth elements. Due to problems such as poor strain adaptability, neglect of interactions between strains, and insufficient stability of the bacterial community, the leaching effect is poor. Artificial synthetic bacterial communities are widely used in various researches due to their advantages such as high efficiency, stability, and controllability, providing new solutions for the green and sustainable development and utilization of ionic rare earth elements. However, there are currently no reports on the application of artificial synthetic bacterial communities in the research of rare earth element bioleaching and its technological advancement. Summary of the invention

[0005] The purpose of the present invention is to provide a method for artificially constructing a synthetic bacterial community with rare earth element leaching function. In view of the shortcomings of the current ionic rare earth element biological leaching technology, the construction method provided by the present invention is based on 16S amplicon high-throughput sequencing technology, combined with microbial pure culture technology, to determine key leaching functional microbial groups and construct artificial leaching functional synthetic bacterial communities. The construction method can make full use of the synergistic effect between strains and improve the adaptability and leaching ability of the bacterial community to complex leaching environment systems.

[0006] To achieve the above object, the present invention provides a method for artificially constructing a synthetic bacterial community with rare earth element leaching function, the method comprising the following steps:

[0007] S1. Enrichment and domestication of rare earth element leaching bacteria: The method of gradually replacing carbon sources with minerals is adopted to enrich and domesticate leaching bacteria that can survive under low nutrient and high mineral concentration conditions from rare earth-rich ore samples;

[0008] S2. Determine the key functional bacterial flora for rare earth element leaching: Use high-throughput sequencing technology to analyze the community diversity and species composition changes of the leaching bacterial flora during the biological leaching of ionic rare earth elements, and determine the key functional bacterial flora in the leaching process;

[0009] S3. Constructing a rare earth element leaching functional strain resource library: separating, screening and identifying leaching functional strains with leaching potential from rare earth-rich ore samples to form a rare earth element leaching functional strain resource library, and determining the leaching ability of the leaching functional strains;

[0010] S4. Screening and determining the initial members of the synthetic bacterial community with leaching function: comparing the ASV sequence information of the key functional bacterial community obtained by high-throughput sequencing with the gene sequence of the leaching functional strain, and then selecting the strain with the highest leaching rate from each species of the leaching functional strain as the initial member based on the principles of high leaching ability and species diversity;

[0011] S5. Optimize the configuration of synthetic bacterial communities with leaching functions: Use the initial members to construct different combinations of synthetic bacterial communities with leaching functions. By analyzing their growth patterns, interactions between strains and leaching capabilities, optimize the configuration of the synthetic bacterial communities and determine the optimal synthetic bacterial community with leaching functions.

[0012] Furthermore, in step S1, the low nutrition and high mineral concentration conditions refer to a carbon source mass percentage of 4.50% and a rare earth mineral mass percentage of 20.50%.

[0013] Further, in step S2, the method for determining the key functional bacterial flora for rare earth element leaching comprises the following specific steps:

[0014] S21, determining the leaching capacity of the leaching flora;

[0015] S22. 16S amplicon high-throughput sequencing technology was used to analyze the community diversity and species composition changes of the leaching flora during the biological leaching of ionic rare earth elements, revealing the succession law of the leaching flora during the biological leaching of rare earth elements;

[0016] S23. Analyze the diversity of bacterial flora: Calculate the species richness and diversity of samples at different time points by calculating the Alpha diversity index of the bacterial flora; Use T-test to evaluate the difference of the Alpha diversity index of the leaching bacterial flora during the leaching process;

[0017] S24. Analyze the microbial community structure: Based on the analysis results of ASV, the community composition of each sample was counted at the phylum and genus classification levels, and the main microbial composition and relative abundance of the dominant flora in each sample at different time points were calculated;

[0018] S25, determining the urease activity of the bacterial flora;

[0019] S26. Identify key functional bacterial communities.

[0020] Further, in step S3, the key functional bacteria are of the genus Bacillus.

[0021] Further, in step S3, the method for constructing a rare earth element leaching functional strain resource library comprises the following specific steps:

[0022] S31. Isolation and purification of leaching functional strains: using separation medium to separate and purify leaching functional strains with leaching potential from rare earth-rich ore samples;

[0023] S32. Phylogenetic analysis of extracted functional strains: The total genomic DNA of bacteria was extracted by CTAB method, and the 16S rRNA gene of the strain was amplified by PCR; the universal primers of bacterial 16S rDNA were used for PCR amplification of 16S rDNA, and the PCR products were sequenced; the sequencing data of bacterial 16S rRNA gene sequences were spliced ​​and compared to obtain species information; and the identification results were verified by constructing a phylogenetic tree;

[0024] S33. Determination of the leaching ability of leaching functional strains.

[0025] Furthermore, the separation medium is selected from one of an organic acid-producing bacteria separation medium, a PKO medium, and a sulfur-oxidizing bacteria separation medium.

[0026] Furthermore, step S4 includes the following specific steps: comparing the ASV sequence information of the key functional flora obtained by high-throughput sequencing with the 16S rRNA gene sequence of the isolated and purified leaching functional strain, sorting the strains with high coverage of the 16S rRNA gene sequence and the ASV sequence information of the key functional flora in the leaching functional strains, and then selecting the strain with the highest leaching rate from each species of the leaching functional strains as the initial member based on the principles of high leaching ability and species diversity.

[0027] Further, in step S4, the initial members are strains B. velezensis GXS-P-3 and B. mucilaginosus GXS-P-4 with phosphate solubilizing ability, strain B. siamensis GXF-A-5 producing organic acid, and strain B. megaterium GXF-S-6 with sulfur oxidation ability.

[0028] Further, in step S5, the specific method for optimizing the configuration of the synthetic bacterial community with leaching function is: constructing different combinations of synthetic bacterial communities with leaching function using equal proportions of initial members, and eliminating strains with inconsistent growth patterns, inhibitory effects on other strains and combinations with low leaching ability by analyzing their growth patterns, interactions between strains and leaching abilities, screening out combinations with synergistic effects between strains, optimizing the configuration of the synthetic bacterial community, and determining the optimal synthetic bacterial community with leaching function.

[0029] Furthermore, based on the differences between the growth curves of single strains and the leaching curves of synthetic bacterial communities, the interactions between strains were analyzed: if there is a synergistic effect between the strains, the OD of the synthetic bacterial community reaching a stable period is greater than that of all its constituent members; if there is an inhibitory effect between the strains, the OD of the synthetic bacterial community reaching a stable period is less than that of one or more of its constituent members.

[0030] The beneficial effects of the present invention are that the method for artificially constructing a synthetic bacterial community with rare earth element leaching function provided by the present invention, by enriching and taming the rare earth element leaching bacterial community, determining the key functional bacterial community of rare earth element leaching, constructing a resource library of rare earth element leaching functional strains, screening and determining the initial members of the synthetic bacterial community with leaching function, and constructing different combinations of synthetic bacterial communities with leaching function in equal proportions using the initial members, by analyzing its growth law, the interaction between strains and the leaching ability, optimizing the configuration of the synthetic bacterial community, and finally obtaining the optimal synthetic bacterial community with leaching function. The method provided by the present invention is based on high-throughput sequencing technology to find key leaching functional microorganisms and give full play to the interaction between strains, improve the adaptability and stability of strains to the stress environment of the leaching system, thereby improving the leaching rate of rare earth metal elements. Moreover, the present invention uses high-throughput sequencing technology and pure culture technology, combined with the concepts of synthetic biology and ecology, to provide a new way for the green and sustainable development and utilization of ionic rare earth elements, and provides a reference for the construction of other metal element leaching functional bacterial communities, which has scientific value and application value. In addition, the microorganisms that make up the bacterial community in the method provided by the present invention all come from the original environment of the mine, and are green and environmentally friendly, have mild conditions, have strong adaptability to the leaching system, and have a good leaching effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic flow chart of a method for artificially constructing a synthetic bacterial community with rare earth element leaching function provided by an embodiment of the present invention;

[0032] Figure 2 It is a schematic diagram of the dynamic change of the leaching rate of the leaching flora bioleaching process provided by the embodiment of the present invention;

[0033] Figure 3It is a schematic diagram of the change of the Alpha diversity index of the microbial community during the leaching process of the leaching flora provided in an embodiment of the present invention;

[0034] Figure 4 It is a schematic diagram of changes in the composition of the microbial community at the genus level during the leaching process of the leaching flora provided by an embodiment of the present invention;

[0035] Figure 5 Schematic diagram of the change of urease activity of leaching flora during the leaching process provided by an embodiment of the present invention;

[0036] Figure 6 It is a ratio diagram of the Bacillus ASVs matched by the initial member strains of the leaching functional synthetic bacterial community provided by the embodiment of the present invention to the total Bacillus ASVs in high-throughput sequencing;

[0037] Figure 7 It is a schematic diagram of the growth rules of synthetic bacterial communities with different leaching functions and initial members provided by an embodiment of the present invention;

[0038] Figure 8 It is a schematic diagram of the leaching rate of rare earth elements by synthetic bacterial communities and initial members with different leaching functions provided by an embodiment of the present invention;

[0039] Fig. 9 It is a schematic diagram of the change of the leaching rate of the rare earth element leaching functional synthetic bacterial community B-2 over time provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0040] The technical solutions in the embodiments of the present invention will be further clearly and completely described below in conjunction with the drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0041] This embodiment aims at the shortcomings of existing biological leaching technology, and aims to provide a method for artificially constructing a synthetic bacterial community with rare earth element leaching function, which enhances the adaptability of strains, the synergistic effect between strains and the leaching capacity. By using high-throughput sequencing technology and pure culture technology, combined with the concepts of synthetic biology and ecology, a new approach is provided for the green and sustainable development and utilization of ionic rare earth elements, and a reference is provided for the construction of other metal element leaching functional bacterial communities, which has certain scientific value and application value. The constituent microorganisms of the bacterial community in this embodiment are all from the original environment of the mine, and have the characteristics of green environmental protection, mild conditions, strong adaptability to the leaching system, and good leaching effect.

[0042] like Figure 1As shown, this embodiment provides a method for artificially constructing a synthetic bacterial community with rare earth element leaching function, the method comprising the following steps:

[0043] S1. Enrichment and domestication of rare earth element leaching bacteria: The method of gradually replacing carbon source with minerals is adopted to enrich and domesticate the leaching bacteria that can survive under low nutrition (carbon source 4.50%) and high mineral concentration (20.50%) conditions from rare earth-rich ore samples.

[0044] S2. Determine the key functional bacterial communities in rare earth element leaching: Use 16S amplicon high-throughput sequencing technology to analyze the community diversity and species composition changes of the leaching bacterial communities during the biological leaching of ionic rare earth elements, revealing the succession law of the leaching bacterial communities during the biological leaching of rare earth elements. At the same time, determine and analyze the key enzyme activities of the leaching bacterial communities to determine the key functional bacterial communities in the leaching process.

[0045] S3. Construct a resource library of rare earth element leaching functional strains: Use organic acid-producing, phosphate-solubilizing and sulfur-oxidizing culture media to separate, screen and identify leaching functional strains with leaching potential from rare earth-rich ore samples, and measure the leaching ability of the obtained strains.

[0046] S4. Screening and determination of the initial members of the synthetic bacterial community with leaching function: The ASV sequence information of the key functional bacterial community obtained by high-throughput sequencing was compared with the 16S rRNA gene sequence of the isolated and purified leaching functional strain, and then based on the principles of high leaching ability and species diversity, the strain with the highest leaching rate was selected from each species of the corresponding strain as the initial member.

[0047] S5. Optimize the configuration of synthetic bacterial communities with leaching functions: Use the initial members in equal proportions to construct synthetic bacterial communities with leaching functions in different combinations. By analyzing their growth patterns, interactions between strains and leaching capabilities, optimize the configuration of the synthetic bacterial communities and ultimately obtain the optimal synthetic bacterial community with leaching functions.

[0048] In this embodiment, the key leaching functional microorganisms are found based on high-throughput sequencing technology and the interaction between strains is fully utilized to improve the adaptability and stability of the strains to the stress environment of the leaching system, thereby improving the leaching rate of rare earth metal elements. The method is applied to the leaching of other metal elements, which can also achieve the effect of enhancing the adaptability of the strains and improving the leaching capacity.

[0049] The technical solution provided by this embodiment is further described in detail below through specific examples.

[0050] Example 1: Enrichment and domestication of rare earth element leaching bacteria

[0051] Weigh 10.0g of rare earth ore, crush it and shake it with 100mL of sterile water (28℃, 180r / min) for 2h, and then let it stand for 30min. Then take 10mL of bacterial suspension (obtain microorganisms from soil with high mineral concentration) and mix it with 90mL LB liquid culture medium, 28℃, speed 140r / min, enrich and culture on a constant temperature shaker for 2-5d. Then, adopt the method of continuous transfer and domestication, first inoculate the enriched leaching flora into the first domestication culture medium for culture, the culture conditions are 28℃, the speed is 140r / min, and culture for 7-10d until the growth of the leaching flora reaches the plateau phase, and then transfer it to the second domestication culture medium for culture, and transfer the culture in turn to finally obtain the target leaching flora. The OD value of the leaching flora is measured every 24h to monitor the changes in the growth of the flora; and the mixed bacterial solution obtained after each domestication is collected, mixed with 30% glycerol at a ratio of 1:1 and stored in a -80℃ refrigerator for subsequent use.

[0052] Among them, the formula of the culture medium used for bacterial colony domestication is: 9K culture medium [formula: (NH 4 ) 2 SO 4 3.0g / L, Na 2 SO 4 2.1g / L, MgSO 4 7H 2 O0.5g / L, K 2 HPO 4 0.05g / L, KCl 0.1g / L and Ca(NO 3 ) 2 0.01g / L, pH 7] as the basis, yeast extract (5.0g) and trypsin (10.0g) were added as energy substances, and 10% of ionic rare earth minerals were added to form the first acclimation medium. The energy substances (1.5g) in the acclimation medium were gradually reduced by 10%, and the mineral concentration was increased by 1.5% accordingly. The details are shown in Table 1 below:

[0053] Table 1 Composition of the culture medium for the nth acclimatization

[0054]

[0055] Note: All culture media need to be sterilized at 121℃ for 20 minutes before use.

[0056] Finally, the mixed bacterial community that can grow under low nutrition (carbon source mass percentage 4.50%) and high rare earth mineral concentration (rare earth mineral mass percentage 20.50%) is the leaching bacterial community. Under the conditions where the carbon source is lower than 4.50% and the rare earth mineral concentration is higher than 20.50%, the bacterial community cannot grow.

[0057] Example 2: Determination of key functional microbial groups for rare earth element leaching

[0058] The method for determining the key functional bacterial flora for rare earth element leaching includes the following specific steps:

[0059] S21. Determine the leaching capacity of the leaching flora: inoculate 2.00% of the leaching flora into a leaching medium containing a mineral concentration of 20.50% and culture for 14 days at a temperature of 28°C and a rotation speed of 140r / min. During this period, samples were taken every 2 days to monitor the changes in the OD value, pH value and rare earth element content of the leaching solution; samples used to monitor the OD value and pH value need to be measured immediately after sampling; and samples used for rare earth content determination need to be centrifuged at room temperature for 10 minutes after collection, and the supernatant is collected, and then filtered with a 0.22μm filter membrane to remove the bacteria and collect the leaching solution, which is stored at 4°C for later use. At the same time, the uninoculated leaching medium is used as a blank control, and the experiment is repeated 3 times.

[0060] S22, 16S amplicon high-throughput sequencing: Microbial samples were collected on days 0.5, 2, 4, 6, 8, 10, 12, and 14 during the leaching process, and the microbial sample on day 0 before leaching was started was used as a control ( Figure 2 CK), a total of 27 microbial samples at 9 time points were used for high-throughput sequencing. Figure 2 The graph shows the dynamic changes of leaching rate during the bioleaching process of the leaching flora.

[0061] S23. Analysis of bacterial community diversity: qiime2 was used to calculate the Alpha diversity index of the bacterial community to calculate the species richness and diversity of samples at different time points. In order to analyze the changes in the diversity of the microbial community during the leaching process, the t-test was used to evaluate the differences in the Alpha diversity index of the leaching bacterial community during the leaching process. The changes in the Alpha diversity index of the microbial community during the leaching process (the Simpson index and Shannon index used to measure species diversity, and the Chao1 index and ACE index used to estimate the unobserved species richness) are shown in the figure. Figure 3 As shown. Figure 3 It can be seen that neither the ACE index nor the Chao1 index changed significantly during the leaching process, that is, the bioleaching process had no effect on the richness of microorganisms in the leaching flora. As the leaching progressed, the Shannon and Simpson indices showed an overall upward trend, that is, the uniformity of the microbial community increased, indicating that the distribution of the microbial community became more uniform as the leaching progressed.

[0062] S24. Analyze the microbial community structure: Based on the analysis results of ASV, the community composition of each sample was statistically analyzed at the phylum and genus classification levels, and the main microbial composition and relative abundance of the dominant flora in each sample at different time points were calculated. In order to explore the succession law of the microbial community during the leaching process and to find the key leaching functional microbial groups, we analyzed the composition and structure of the leaching microorganisms and the differences and changes in the abundance of the dominant flora from a time gradient. The changes in the composition of the microbial community at the genus level during the leaching process are as follows: Figure 4 As shown. Figure 4 It can be seen that from the overall leaching process, the main dominant group at the genus level is Sporosarcina, and its relative abundance decreased from 99.81±0.03% to 98.40±0.03%; Bacillus underwent significant changes, and its relative abundance increased from 0.02±0.01% to 1.48±0.23% as the leaching progressed.

[0063] S25. Determine the urease activity of the bacterial flora: collect 5 mL of bacterial solution every 2 days, then take 2 mL of bacterial solution and mix it with 18 mL of 1.1 mol / L urea solution in a 50 mL centrifuge tube. Use a conductivity meter to measure the conductivity of the solution every minute within 5 minutes. The average conductivity change value (mS / cm / min) of the solution within 5 minutes multiplied by the dilution factor of the bacterial solution is the urease activity (mmol / L / min) of the bacterial solution to be tested. The change of urease activity of the leaching flora during the leaching process is as follows: Figure 5 As shown by Figure 5 It can be seen that the main function of the dominant genus (Sporosarcina) in the leaching flora is to precipitate rare earth elements.

[0064] S26. Determine the key functional microbial groups: By analyzing the community composition and diversity of the leaching flora during the leaching process, it was found that the diversity of the leaching flora did not change significantly, but the uniformity gradually increased; the composition of the leaching flora did not change significantly at the phylum level, but at the genus level, the relative abundance of Bacillus gradually increased from 0.02±0.01% to 1.48±0.23% with the increase of the leaching rate (e.g. Figure 4 Combined with the determination and analysis of the urease activity of the leached bacterial community, it was determined that the key functional microbial group (i.e., the key functional bacterial community) was Bacillus.

[0065] Example 3: Construction of a rare earth element leaching functional strain resource library

[0066] The method for constructing a rare earth element leaching functional strain resource library comprises the following specific steps:

[0067] S31. Isolation and purification of leaching functional strains: Weigh 10.0g of ionic rare earth mineral sample rich in rare earth elements and add it to a 500mL conical flask containing 100mL sterile water (containing a small amount of sterilized glass beads), put it into a constant temperature shaker at 150r / min and 28℃ for 1.5-2h → after standing for 15min, take 10mL of bacterial solution (supernatant) and enrich it in different separation (liquid) culture media for 48h; in order to avoid excessive or insufficient growth of strains on some culture media, the bacterial solution was graded diluted and applied to the culture medium (500μL+4.5mL sterile water, from 10 -1 Dilute to 10 -7 ), and cultured in a constant temperature incubator at 28°C; each plate was observed every 12 hours. If a single colony appeared, the single colony was inoculated into a new culture medium with an inoculation loop for purification. The pure culture strain was purified at least three times, and then the pure culture bacteria were stored in a 30% (v / v) concentration of glycerol solution and stored in a -80°C ultra-low temperature refrigerator for subsequent experiments.

[0068] In a specific embodiment, the rare earth element-rich ionic rare earth ore sample was collected from Dayangkeng mining area, Dabu Township, Gan County, Ganzhou City, located in the southern part of Jiangxi Province (25°46'51.6"N, 114°56'

[0069] 56.5"E), the main rare earth elements in the mineral samples are shown in Table 2 below, which belong to ionic light rare earth ore; this type of rare earth ore is mainly distributed in Heling Mining Area, Nanqiao Mining Area, Xunwu County, Ganzhou City and Shangwentan Mining Area in Gan County, among which the common microorganisms are Bacillus, Paenibacillus, Pseudomonas, Brevundimonas and Delftia.

[0070] Table 2 Percentage of each element in rare earth ore samples

[0071]

[0072] In step S31, the separation medium is selected from one of an organic acid-producing bacteria separation medium, a PKO medium, and a sulfur-oxidizing bacteria separation medium, and the compositions of the three separation mediums are respectively as follows:

[0073] (1) Organic acid-producing bacteria isolation medium: 5 g of peptone, 10 g of glucose, 10 g of yeast extract (dissolved in hot water), 4 mL of 1.6% (w / v) bromocresol purple indicator (acid-base indicator), 20 g of agar, and the pH value is adjusted to 6.0-6.2.

[0074] (2) PKO medium (phosphate-solubilizing bacteria solid medium): 5.0 g Ca 3 (PO 4 ) 2 , 10.0 g glucose, 0.1 g (NH 4 ) 2 SO 4 , 0.5g NaCl, 0.2g KCl, 0.1g MgSO 4 7H 2 O, 0.002 g FeSO 4 , 0.002g MnSO 4 , 0.5g yeast extract, 20g agar, pH value is 7.0-7.5.

[0075] (3) Sulfur oxidizing bacteria isolation medium: (1) Na 2 S 2 O 3 ·5H 2 O 8g (easy to oxidize at high temperature, so it needs to be filtered and sterilized through a 0.22μm filter membrane), K 2 HPO 4 1.2g, KH 2 PO 4 1.2g, NH 4 Cl·6H 2 O 0.4 g, MgCl 2 6H 2 O 0.2g, MgSO 4 7H 2 O 0.01g, agar 20g, pH value is 7.0-7.2.

[0076] S32, Phylogenetic analysis of extracted functional strains: The total genomic DNA of bacteria was extracted by CTAB method, and the 16S rRNA gene of strains was amplified by PCR. The universal primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r were used to amplify the 16S rRNA gene of bacteria.

[0077] PCR amplification of 16S rDNA (5′-GGTTACCTTGTTACGACTT-3′) was performed. PCR reaction system (50 μL): DNA 2 μL, 10× Buffer 5 μL, MgCl 2(25mmoL / L) 3μL, dNTPs (10mmoL / L) 1μL, primers 27F and 1492R (10μmoL / L) 1μL each, Taq enzyme (5U / μL) 0.5μL. PCR reaction conditions: 94℃5min; 94℃1min, 55℃1min, 72℃2min, 35 cycles; 72℃5min. The PCR product was sent to the sequencing company for sequencing. The 16S rRNA gene sequence data of the bacteria were spliced ​​on the Seqman software and saved as fasta files. The EzbioCloud website (https: / / www.ezbiocloud.net / ) was used for comparison to obtain species information. The identification results were then verified by constructing a phylogenetic tree. The sequences obtained by sequencing and the homologous series obtained by BLAST were compared using the ClustalW program in the MEGA 8.0 software. The similarity of each pair of similar sequences was calculated using DNAMAN 8.0 software (Lynnon Biosoft, USA).

[0078] S33. Determination of the leaching ability of leaching functional strains: inoculate 2.00% (v / v) bacterial liquid into the leaching medium (the mineral concentration of the leaching medium is 2.00% (w / v)) (the mineral is ground and passed through a 60-mesh sieve); place it on a constant temperature shaker at 28°C and 140rpm for 7 days. Collect 5mL of the supernatant of the leaching solution on the last day, centrifuge it at 6000g for 10 minutes at room temperature, then filter it with a 0.22μm filter membrane to collect the leaching solution, and store it in a refrigerator at 4°C for testing. Use the arsenazo (III) colorimetric method to determine the content of rare earth elements in the leaching solution, and then calculate the leaching rate of rare earth elements for each strain. The leaching rate is the overall leaching rate of all rare earth elements in the rare earth ore sample.

[0079] The specific method of the arsenazo (III) colorimetric method is as follows: the raw ore used for leaching is fully leached with 2% ammonium sulfate, the leachate is precipitated with oxalic acid and calcined to obtain a mixed rare earth oxide, the rare earth oxide is accurately weighed, dissolved with hydrochloric acid and the pH value is adjusted to 5.00, and the rare earth standard solution is obtained after constant volume. 10μL, 25μL, 50μL, 100μL, 150μL, 200μL, and 300μL of 0.2g / L rare earth standard solution are taken in a 25mL colorimetric tube respectively, 5ml of acetic acid-ammonium acetate buffer solution with a pH value of 3.33 is added, and 2.5mL of azoarsenic (III) solution with a concentration of 0.2g / L is added, and after shaking and standing for 30min, the absorbance at a wavelength of 655nm is measured, and a standard curve is drawn. The absorbance of the leachate is measured according to the above method, and its corresponding concentration is obtained in the standard curve. The culture medium without bacteria is used as a blank control, and each group of experiments is repeated 3 times.

[0080] Example 4: Construction of synthetic bacterial flora with rare earth element leaching function

[0081] The method for constructing a synthetic bacterial community with rare earth element leaching function comprises the following specific steps:

[0082] S41, screening and determining the initial members of the synthetic bacterial community with leaching function: the ASV sequence information of the key functional bacterial community obtained by high-throughput sequencing was compared with the 16S rRNA gene sequence of the isolated and purified leaching functional strain, and the strains with high coverage of the 16S rRNA gene sequence and the ASV sequence information of the key functional bacterial community were sorted, and then based on the principle of high leaching ability and species diversity (high ASV coverage of the key functional bacterial community), the strain with the highest leaching rate was selected from each species of the leaching functional strain as the initial member. The information and leaching rate of the selected initial members of the synthetic bacterial community with leaching function are shown in Table 2, which are the strains B. velezensis GXS-P-3 and B. mucilaginosus GXS-P-4 with phosphate solubilization ability, the strain B. siamensis GXF-A-5 producing organic acid, and the strain B. megaterium GXF-S-6 with sulfur oxidation ability.

[0083] Table 3 Information and leaching rates of initial members of the leaching functional synthetic bacterial consortium

[0084]

[0085]

[0086] The ratio of Bacillus ASVs matched by the initial members of the leached functional synthetic bacterial consortium to the total Bacillus ASVs in high-throughput sequencing is as follows: Figure 6 shown.

[0087] S42. Optimize the configuration of synthetic bacterial flora with leaching function: Use these four strains in equal proportions to construct different combinations of synthetic bacterial flora with leaching function, as shown in Table 3. By analyzing their growth patterns, interactions between strains and leaching abilities, strains with inconsistent growth patterns, inhibitory effects on other strains and combinations with low leaching abilities are eliminated.

[0088] Specifically, based on the difference between the growth curve of a single strain and the leaching curve of the synthetic bacterial consortium, the interaction between the strains was analyzed. If there is a synergistic effect between the strains, the OD of the synthetic bacterial consortium reaching the stable period is greater than that of all its component members; if there is an inhibitory effect between the strains, the OD of the synthetic bacterial consortium reaching the stable period is less than one or more of its component members.

[0089] The growth curves of synthetic bacterial communities with different leaching functions and initial members are as follows: Figure 7As shown in Figure 2, the leaching rates of rare earth elements by synthetic bacterial consortia with different leaching functions and initial members are as follows: Figure 8 shown.

[0090] Table 4 Composition and number of synthetic bacterial consortia with different leaching functions

[0091]

[0092]

[0093] S43, determine the optimal leaching function synthetic bacterial community: based on the principle of strong leaching ability, mutual coordination of strains, and consistent growth rules, the optimal configuration of the leaching function synthetic bacterial community is finally obtained. The optimal configuration is the rare earth element leaching function synthetic bacterial community B-2, whose constituent strains are B.siamensis GXF-A-5, B.velezensis GXS-P-3, and B.mucilaginosus GXS-P-4. Among these three strains, B.siamensis GXF-A-5 has the ability to produce organic acids, while B.velezensis GXS-P-3 and B.mucilaginosus GXS-P-4 have the ability to solubilize phosphorus. The above three strains are mixed in equal proportions to construct a synthetic bacterial community, which is a leaching function synthetic bacterial community. Under the conditions of a mineral concentration of 2.00%, a bacterial community inoculation amount of 2.00%, and an initial pH value of 6.7 in the leaching solution, the leaching rate is 46.72±1.87%. Schematic diagram of the change of leaching rate of rare earth element leaching functional synthetic bacteria group B-2 over time Fig. 9 shown.

[0094] Compared with a single strain in its composition, the synthetic bacterial community with rare earth element leaching function artificially constructed according to the above method fully utilizes the synergistic effect between strains, enhances the adaptability of strains, and thus significantly improves the leaching rate of rare earth elements, showing a good leaching effect. Therefore, the method of the present invention provides a new way for the green and sustainable development and utilization of ionic rare earth elements.

[0095] The above only expresses the implementation mode of the present invention, and its description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, without departing from the concept of the present invention, a number of modifications, improvements and substitutions are made to achieve the technical effects of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. A method for artificially constructing a synthetic bacterial community with rare earth element leaching function, characterized in that: The method comprises the following steps: S1. Enrichment and domestication of rare earth element leaching bacteria: The method of gradually replacing carbon sources with minerals is adopted to enrich and domesticate leaching bacteria that can survive under low nutrient and high mineral concentration conditions from rare earth-rich ore samples; S2. Determine the key functional bacterial flora for rare earth element leaching: Use high-throughput sequencing technology to analyze the community diversity and species composition changes of the leaching bacterial flora during the biological leaching of ionic rare earth elements, and determine the key functional bacterial flora in the leaching process; S3. Constructing a rare earth element leaching functional strain resource library: separating, screening and identifying leaching functional strains with leaching potential from rare earth-rich ore samples to form a rare earth element leaching functional strain resource library, and determining the leaching ability of the leaching functional strains; S4. Screening and determining the initial members of the synthetic bacterial community with leaching function: comparing the ASV sequence information of the key functional bacterial community obtained by high-throughput sequencing with the gene sequence of the leaching functional strain, and then selecting the strain with the highest leaching rate from each species of the leaching functional strain as the initial member based on the principles of high leaching ability and species diversity; S5. Optimize the configuration of synthetic bacterial communities with leaching functions: Use the initial members to construct different combinations of synthetic bacterial communities with leaching functions. By analyzing their growth patterns, interactions between strains and leaching capabilities, optimize the configuration of the synthetic bacterial communities and determine the optimal synthetic bacterial community with leaching functions.

2. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 1, characterized in that: In step S1, the low nutrition and high mineral concentration conditions refer to a carbon source mass percentage of 4.50% and a rare earth mineral mass percentage of 20.50%.

3. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 2, characterized in that: In step S2, the method for determining the key functional bacterial flora for rare earth element leaching comprises the following specific steps: S21, determining the leaching capacity of the leaching flora; S22. 16S amplicon high-throughput sequencing technology was used to analyze the community diversity and species composition changes of the leaching flora during the biological leaching of ionic rare earth elements, revealing the succession law of the leaching flora during the biological leaching of rare earth elements; S23. Analyze the diversity of bacterial flora: Calculate the species richness and diversity of samples at different time points by calculating the Alpha diversity index of the bacterial flora; Use t-test to evaluate the difference of the Alpha diversity index of the leaching bacterial flora during the leaching process; S24. Analyze the microbial community structure: Based on the analysis results of ASV, the community composition of each sample was counted at the phylum and genus classification levels, and the main microbial composition and relative abundance of the dominant flora in each sample at different time points were calculated; S25, determining the urease activity of the bacterial flora; S26. Identify key functional bacterial communities.

4. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 3, characterized in that: In step S3, the key functional bacteria are of the genus Bacillus.

5. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 2, characterized in that: In step S3, the method for constructing a rare earth element leaching functional strain resource library comprises the following specific steps: S31. Isolation and purification of leaching functional strains: using separation medium to separate and purify leaching functional strains with leaching potential from rare earth-rich ore samples; S32. Phylogenetic analysis of extracted functional strains: The total genomic DNA of bacteria was extracted by CTAB method, and the 16S rRNA gene of the strain was amplified by PCR; the universal primers of bacterial 16S rDNA were used for PCR amplification of 16S rDNA, and the PCR products were sequenced; the sequencing data of bacterial 16S rRNA gene sequences were spliced ​​and compared to obtain species information; and the identification results were verified by constructing a phylogenetic tree; S33. Determination of the leaching ability of leaching functional strains.

6. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 5, characterized in that: The separation medium is selected from one of an organic acid-producing bacteria separation medium, a PKO medium, and a sulfur-oxidizing bacteria separation medium.

7. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 2, characterized in that: Step S4 includes the following specific steps: comparing the ASV sequence information of the key functional flora obtained by high-throughput sequencing with the 16S rRNA gene sequence of the isolated and purified leaching functional strain, sorting the strains with high coverage of the 16S rRNA gene sequence and the ASV sequence information of the key functional flora in the leaching functional strains, and then selecting the strain with the highest leaching rate from each species of the leaching functional strains as the initial member based on the principles of high leaching ability and species diversity.

8. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 7, characterized in that: In step S4, the initial members are strains B. velezensis GXS-P-3 and B. mucilaginosus GXS-P-4 with phosphate solubilizing ability, strain B. siamensis GXF-A-5 producing organic acid, and strain B. megaterium GXF-S-6 with sulfur oxidation ability.

9. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 7, characterized in that: In step S5, the specific method for optimizing the configuration of the synthetic bacterial community with leaching function is: constructing different combinations of synthetic bacterial communities with leaching function using equal proportions of initial members, and eliminating strains with inconsistent growth patterns, inhibitory effects on other strains, and combinations with low leaching abilities by analyzing their growth patterns, interactions between strains, and leaching abilities, screening out combinations with synergistic effects between strains, optimizing the configuration of the synthetic bacterial community, and determining the optimal synthetic bacterial community with leaching abilities.

10. The method for artificially constructing a synthetic bacterial community with rare earth element leaching function according to claim 9, characterized in that: Based on the differences between the growth curves of single strains and the leaching curves of synthetic bacterial communities, the interactions between strains were analyzed: if there was a synergistic effect between the strains, the OD of the synthetic bacterial community reaching a stable period would be greater than that of all its constituent members; if there was an inhibitory effect between the strains, the OD of the synthetic bacterial community reaching a stable period would be less than that of one or more of its constituent members.

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