Strain for enriching lithium in salt lake brine and application thereof

Abstract

This invention provides a strain of the genus *Psychrophilus* (…). Psychrobacter alimentarius The strain WXL2025-01, with accession number CGMCC No. 37430 at the China General Microbiological Culture Collection Center, exhibits good tolerance to high salt concentrations and can grow in a medium containing 90 g / L NaCl. Under low-temperature conditions (10 °C), it can enrich Li elements in brine containing a complex ionic environment within 15 days, achieving an enrichment rate of 22%. This invention also provides the culture medium and cultivation method for this strain. This invention provides a core superior strain for the effective enrichment of Li elements in brine, increasing specificity, saving resources, and eliminating secondary pollution, making it suitable for large-scale fermentation production.

Description

Technical Field

[0001] This invention relates to the field of applied microbiology, specifically to a strain of microorganism capable of enriching lithium in salt lake brine and its applications. Background Technology

[0002] Salt lakes, as "liquid mines," are important reservoirs of extreme environments and strategic resources. In China's modern salt lakes, the brine is rich in inorganic salt resources. In addition to elements such as potassium, sodium, chlorine, and magnesium, the total reserves of lithium, boron, rubidium, cesium, iodine, and bromine are also considerable, with a potential economic value exceeding 80 trillion yuan.

[0003] Salt lakes possess valuable elements, but these resources are abundant in total quantity but low in content, with complex associated ion types, making extraction difficult or prohibitively expensive. Chemical companies typically extract the thickest layers of salt lakes while discarding the thinner ones, focusing on high-quality elements and neglecting lower-quality ones. This prevents the complete utilization of valuable elements, resulting in many being discarded as tailings due to low concentrations and insufficient grade. This includes lithium (Li), an element with very high commercial value. This not only causes serious resource waste but also environmental pollution.

[0004] The lithium element in the brine exists in an ionic state (Li). + ), and with other elements (Na) + K + Lithium (such as lithium, Mg, Mg, and Mg) has similar chemical properties, making separation and purification difficult. Traditional methods for lithium separation mainly include physical and chemical methods, such as extraction, acidification, adsorption, flotation, and crystallization. Physical and chemical methods suffer from many drawbacks, including poor specificity, low extraction rates, and high costs, which limit their practical application.

[0005] Microbial enrichment is a highly specific, efficient, environmentally friendly, and cost-effective method for enriching valuable elements. It offers advantages such as large enrichment capacity and high removal efficiency.

[0006] This study screened a strain capable of enriching lithium and investigated its enrichment performance, providing a new technology for the enrichment, separation, and purification of valuable elements in salt lakes and improving the utilization rate of salt lake resources. Summary of the Invention

[0007] The purpose of this invention is to provide a technical solution that overcomes the shortcomings of the prior art, namely, to provide a strain capable of specifically enriching ionic lithium and its application.

[0008] The objective of this invention is to provide a strain of the genus *Psychrophilus*. Psychrobacter alimentarius (WXL2025-01) (hereinafter referred to as WXL2025-01) WXL2025-01The WXL2025-01 strain has the accession number CGMCC No. 37430 at the China General Microbiological Culture Collection Center.

[0009] The characteristic of WXL2025-01 is that this strain can enrich Li in salt lake brine. + WXL2025-01 is a Gram-negative bacterium, CAT-positive, with a maximum tolerance concentration of 90 g / L for NaCl. At 10°C and an OD600 value of 0.1, it can reduce the concentration of Li in a 1.00 mg / L salt lake brine to a concentration of 1.00 mg / L after 15 days. + Enrichment was performed to reduce the concentration of Li to 0.78 mg / L, achieving a removal rate (enrichment rate) of 22%. After centrifugation, the bacterial cells were precipitated from the brine, dried, and weighed. The cells were then digested using a concentrated nitric acid-hydrogen peroxide system, and the enrichment capacity for Li was determined to be 7.10 mg / g dry bacterial weight. The selectivity coefficient (Li / K) of this bacterium reached 10.92, indicating high specificity.

[0010] The second objective of this invention is to provide WXL2025-01 Culture medium and conditions: Culture medium: 8 g / L beef extract, 20 g / L peptone, 80 g / L NaCl, 30 g / L agar. Incubate at 28 ℃; pH adjustment is not required. Growth occurs in approximately 5 days.

[0011] Another object of the present invention is to provide strain WXL2025-01 and its application in lithium (Li) enrichment, specifically to provide lithium (Li) enrichment products using strain WXL2025-01.

[0012] Compared with the prior art, the present invention has the following advantages and effects: the WXL2025-01 strain has good tolerance to high salt concentration (90g / L NaCl concentration) and can carry out lithium (Li) enrichment treatment at low temperature (10 ℃); the WXL2025-01 strain is a bacterium, which does not require additional chemical reagents during the enrichment process, does not produce secondary pollution, and is environmentally friendly; the determination of the dedicated culture medium and culture conditions ensures the excellent proliferation and subculturing of the WXL2025-01 strain, which can be used for large-scale fermentation production and long-term use, and has a certain degree of selectivity, reducing the probability of contamination by other bacteria, reducing costs, and facilitating operation and maintenance.

[0013] Preservation instructions: Bacterial strain name: Psychrophilic bacteria Latin name: Psychrobacter alimentarius Strain number: WXL2025-01 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: January 19, 2026 CGMCC Registration Number: 37430 WXL2025-01 sequencing results: The sequence is a 16S ribosomal RNA gene, 1406 bp in length, as shown in the sequence listing appendix. NCBI database BLAST results: Psychrobacter alimentarius (JG-100), partial sequence ident=99.86%. Attached Figure Description

[0014] Figure 1 This is a colony photograph of strain WXL2025-01 obtained in Example 1. Detailed Implementation

[0015] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0016] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0017] The culture media used in the following examples are as follows: Culture medium: 8 g / L beef extract, 20 g / L peptone, 80 g / L NaCl, 30 g / L agar. Incubate at 28 ℃; pH adjustment is not required. Growth complete in approximately 5 days.

[0018] Example 1: Separation and Identification Experiment of WXL2025-01 Prepare the culture medium, sterilize at 120℃ for 30 min, and cool in petri dishes to form plates. Dilute saline lake water samples and activated sludge from the bottom of salt fields with sterile water, spread them on culture plates, and incubate at 28℃ in the dark. Perform streak plating to separate and purify the culture to obtain WXL2025-01.

[0019] The strain WXL2025-01, isolated and purified using the above steps, was used to extract total DNA as a template for gene amplification. PCR was performed using universal primers for bacterial 16S rDNA on a PCR instrument. After the reaction, 2 μL of the PCR product was analyzed by 1% agarose gel electrophoresis to confirm the PCR amplification fragments. The PCR products were recovered using the AxyPrep DNA gel recovery kit, following the kit's instructions. The purified PCR products from each bacterial species were sequenced using an ABI3730-XL sequencer. Homology comparison with known sequences in NCBI GenBank was performed to determine the strain species and classify them into genus or species.

[0020] PCR amplification yielded a 16S rDNA fragment of approximately 1.4 kb from strain WXL2025-01. After sequence determination, online homology comparison was performed with publicly available 16S rDNA sequences in the NCBI database. The results showed that WXL2025-01 was homologous to... Psychrobacter alimentarius (JG-100) has the highest homology, reaching 99.86%.

[0021] Example 2: Enrichment experiment of lithium (Li) by WXL2025-01 Culture medium: 8 g / L beef extract, 20 g / L peptone, 80 g / L NaCl. The experiment was conducted as follows: After sterilizing the culture medium at high temperature and allowing it to cool to room temperature, it was inoculated with strain WXL2025-01. The medium was cultured at 28℃ for approximately 5 days. The OD600 value of the culture solution was measured using a spectrophotometer. The culture endpoint was reached when the OD600 value was approximately 0.2. The liquid culture medium was centrifuged at 4000 rpm for 5 minutes, and the bacterial cells at the bottom of the centrifuge tube were collected for later use. A certain amount of brine from the salt lake was diluted until the lithium (Li) concentration in the solution was close to 1 mg / L. A microbial enrichment experiment was conducted, with two groups: a microbial treatment group (where the collected bacterial cells were added to the brine and mixed thoroughly until the OD600 value was close to 0.1, at which point the addition was stopped, and the brine was placed at a constant temperature of 10℃ for static treatment); and a blank control group (where the same procedures as the treatment group were followed, except that the patented strain was not inoculated). Two groups were sampled simultaneously at days 0, 5, 10, and 15. The collected samples were centrifuged at 4000 rpm for 5 minutes, and the supernatant was collected and stored at 4℃ for testing. After day 15, the lithium (Li) concentration in the supernatant was determined. The lithium (Li) concentration was measured using atomic absorption spectrophotometry. The results are as follows: Table 1. Changes in lithium (Li) elemental concentration

[0022] Blank control group 0.99 0.99 0.99 0.99 Microbial treatment group 0.99 0.94 0.85 0.78 This experiment demonstrates that: 1. WXL2025-01 has the ability to enrich lithium (Li) in salt lake brine. 2. Under high salinity (the salt lake brine is highly saline and close to saturation), at 10℃, and with a cell concentration of 0.1 at an OD600 value, WXL2025-01 can remove 1.0 mg / L of lithium (Li) to 0.78 mg / L within 15 days, with a Li removal rate (enrichment rate) of 22%.

[0023] Example 3: Determination of Lithium (Li) Enrichment in WXL2025-01 The lithium (Li) enriched bacterial cells obtained by centrifugation in Example 2 were dried at 105 °C to constant weight, and the cell weight was measured to obtain the dry weight (g). The dried bacterial cells were then subjected to microwave digestion (concentrated nitric acid + hydrogen peroxide system). The digestion solution was diluted to volume with 1% nitric acid and the volume (L) was recorded. The concentration of lithium (Li) in the digestion solution was detected using the atomic absorption spectrophotometry method described in Example 2, and the enrichment capacity was calculated. Enrichment capacity (unit: mg Li / g dry bacterial cell weight), calculation formula: Enrichment amount q (mg / g) = (C * fixed volume L) / dry weight of bacterial cells In the formula: C corresponds to the mass concentration of lithium (Li) in the digestion solution, in mg / L.

[0024] Results: The enrichment capacity of this bacterium for Li was 7.10 mg / g dry bacterial weight.

[0025] Example 4: Determination of Selectivity Coefficient The enrichment experiment of potassium (K) by WXL2025-01 was established using the same method as in Example 2, and the enrichment amount of potassium (K) by WXL2025-01 was determined using the method in Example 3. Selectivity coefficient (S) Li / K Calculation formula: S Li / K = Enrichment (Li) / Enrichment (K).

[0026] result: Table 2. Changes in potassium (K) concentration

[0027] Blank control group 1.00 0.99 0.99 0.99 Microbial treatment group 1.00 0.99 0.99 0.98 The WXL2025-01 strain can reduce potassium levels in salt lake brine from an initial concentration of 1 mg / L to a concentration of 1 mg / L within 15 days. + This reduced the concentration to 0.980 mg / L, resulting in a K removal rate (enrichment rate) of 2%. The K enrichment capacity was 0.02 mg / 0.031 g = 0.65 mg / g dry bacterial weight. The selectivity coefficient (S...) Li / K The answer is: 7.10 / 0.65 = 10.92.

[0028] Example 5: Salt tolerance experiment of strain WXL2025-01 To verify the salt tolerance of the strain of this invention, a series of high-salt culture media with varying concentrations were prepared. The culture media formulations were as follows: 8 g / L beef extract, 20 g / L peptone, 30 g / L agar, and NaCl, with NaCl concentrations set as a gradient: 30, 50, 70, 80, 85, 90, 95, and 100 g / L. The media were sterilized at 120°C for 30 min and cooled in petri dishes to form plates. Colonies of the strain were picked from pure cultures, spread, and incubated at 28°C. After 7 days, the culture results were examined, and a positive result was defined as the formation of proliferating colonies.

[0029] Results: WXL2025-01 could grow normally and show positive results on culture media with NaCl concentrations of 30-90 g / L. However, it grew slowly or not at higher NaCl concentrations, such as 95 or 100 g / L. The maximum salt tolerance concentration of this strain was determined to be 90 g / L.

Claims

1. A strain of psychrophilic bacillus ( ) Psychrobacter alimentarius The strain WXL2025-01 has the accession number CGMCC No.37430 at the China General Microbiological Culture Collection Center.

2. The *Psychrophilic Bacillus* strain WXL2025-01 according to claim 1, characterized in that... This strain can accumulate lithium.

3. The *Psychrophilic Bacillus* strain WXL2025-01 according to claim 1, characterized in that... This strain achieved a lithium removal or enrichment rate of 22%.

4. The *Psychrophilus* strain WXL2025-01 according to claim 1, characterized in that... This strain enriched 22% of the lithium in the salt lake brine within 15 days at 10℃ and a cell concentration of 0.1 OD600.

5. A culture medium for culturing the strain of claim 1, characterized in that... The formula is: 8 g / L beef extract, 20 g / L peptone, and 80 g / L NaCl.

6. A method for cultivating the salt tolerance of the strain of claim 1, characterized in that... It can grow normally at a NaCl concentration of 90 g / L.

7. A product for the microbial enrichment of lithium in brine, characterized in that... The product is enriched using the *Cryptospirobacter* strain WXL2025-01 as the core bacterial agent as described in claim 1.

Images