Microbacterium and application thereof in enrichment and separation of lithium in low-quality brine

Abstract

The invention belongs to the technical field of microorganisms and environmental protection, and particularly relates to a microbacterium strain and application thereof in enrichment and separation of lithium in low-quality brine. The strain is preserved in the China Center for Type Culture Collection on February 17, 2025, and the preservation number is CCTCC NO: M 2025230. Experiments prove that the microbacterium can specifically and selectively adsorb and enrich a large amount of lithium from typical low-quality brine thick oil produced water, and meanwhile, the strain is screened from an extreme environment, so that the strain has good tolerance to environmental stress such as high and low temperature, salinity and the like; the method can be used for lithium enrichment and separation of low-quality brine, especially thick oil produced water, and has practical application value for sustainable and eco-friendly development of the lithium industry.

Description

Technical Field

[0001] The present invention belongs to the technical fields of microorganisms and environmental protection, and particularly relates to a Microbacterium strain and its application in the enrichment and separation of lithium from low-quality brine. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention and does not necessarily constitute an admission or imply in any form that this information forms the prior art already known to those of ordinary skill in the art.

[0003] Lithium is a metal widely regarded as having high electrochemical activity and is a strategic metal of great significance in the 21st century and a new energy metal with broad prospects. The rise of the new energy industry has led to a significant increase in the demand for lithium batteries. The supply-demand contradiction of lithium resources has caused the price of lithium to rise rapidly. In addition to powering electric vehicles, lithium-ion batteries are also crucial for grid energy storage related to renewable energy such as wind and solar energy, thereby accelerating carbon neutrality and reducing carbon emissions. Predictions show that from 2023 to 2050, lithium demand will increase by 8 to 10 times. Therefore, the extraction of lithium resources through unconventional channels has also attracted people's attention. Low-quality brine is an important potential source of lithium, with rich reserves and wide geographical distribution. Oilfield produced water is a typical representative of brine, and there is potential for recovering lithium from heavy oil produced water, which may help solve the problem of the imbalance between the supply and demand of lithium resources.

[0004] The enrichment and recovery of useful metals from low-quality brine by special microorganisms have always been a hot topic. Compared with high-energy-consuming and by-product-producing aqueous lithium separation methods such as solvent extraction and ion exchange, using bacteria to extract lithium is considered a feasible, non-toxic, low-cost method and an effective alternative. One of the significant advantages of microbial processes is their ability to operate under mild conditions, which effectively simplifies downstream processing. In some cases, special bacteria can specifically adsorb and enrich lithium from various other ions in produced water through their metabolic activities to produce abundant extracellular polymers. These special microorganisms are usually extremophiles that exist and grow in environments with extreme temperatures, extreme pH conditions, or high salinity. This extraordinary adaptability enables them to play a role in heavy oil produced water. However, so far, there have been few reports on screening special strains with lithium-selective enrichment functions from extreme environments for the recovery of lithium from low-quality brine. Summary of the Invention

[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides a strain of Microbacterium and its application in the enrichment and separation of lithium from low-quality brine. Through experiments, it has been proven that this Microbacterium can specifically and selectively adsorb and enrich a large amount of lithium from produced water of heavy oil. At the same time, since it is screened from extreme environments, this strain has good tolerance to environmental stresses such as high and low temperatures and salinity, and can be used for the enrichment and separation of lithium from low-quality brine, especially produced water of heavy oil. Based on the above research results, the present invention is thus completed.

[0006] The present invention is achieved through the following technical solutions:

[0007] In the first aspect of the present invention, a strain of Microbacterium is provided, named Microbacterium paraoxydans HB04. This strain was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on February 17, 2025, and the deposit number is CCTCC NO: M 2025230.

[0008] In the second aspect of the present invention, a microbial inoculum is provided, which contains the Microbacterium described in the first aspect above, or its fermented product, or its metabolite.

[0009] In the third aspect of the present invention, the application of the above-mentioned Microbacterium or the above-mentioned microbial inoculum as a lithium enrichment agent is provided.

[0010] In the fourth aspect of the present invention, the application of the above-mentioned Microbacterium or the above-mentioned microbial inoculum in the selective enrichment and separation of lithium in a lithium-containing environment is provided.

[0011] Since the Microbacterium in the present invention can grow and reproduce at 10 - 50 °C, pH 4.0 - 11.0, and salinity 0 - 7%, and can specifically enrich lithium to a certain extent, it can adapt to a variety of harsh lithium-containing environments, such as high or low temperature environments, acidic or alkaline environments, low salt or high salt environments.

[0012] In the fifth aspect of the present invention, a method for enriching and separating lithium from low-quality lithium-containing brine is provided. The method includes: applying the above-mentioned Microbacterium or the above-mentioned microbial inoculum to the low-quality lithium-containing brine.

[0013] Specifically, the low-quality lithium-containing brine can be produced water from oilfields, especially produced water of heavy oil.

[0014] The beneficial effects of the above one or more technical solutions:

[0015] The above technical solution provides a strain of Microbacterium, which can adapt to a wide range of temperatures, pH values, and salinities, can grow and reproduce at 10-50 °C, pH 4.0-11.0, and salinity 0-7%, and can specifically enrich lithium to a certain extent. Moreover, at 10-40 °C, pH 5.0-8.0, and salinity 0-5%, the lithium accumulation amount and enrichment rate hardly decay.

[0016] In summary, the above technical solution realizes the efficient recovery of lithium in low-quality brine through the microbial method, has the advantages of simple operation, environmental friendliness, strong adaptability, etc., and provides an innovative solution for the sustainable development of lithium resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings forming a part of this invention are used to provide a further understanding of the invention. The schematic embodiments and descriptions thereof of the invention are used to explain the invention and do not constitute an improper limitation of the invention. Figure 1 It is a colony plate culture photograph of the strain HB04 of the present invention; among them, A is the complete plate culture photograph, and B is the partial plate culture photograph.

[0018] Figure 2 It is an optical microscope photograph of the strain HB04 of the present invention.

[0019] Figure 3 It is a phylogenetic tree of the strain HB04 of the present invention.

[0020] Figure 4 It is the growth curve (A) and lithium enrichment ability (B) of the strain HB04 of the present invention at different initial Li + concentrations.

[0021] Figure 5 It is the growth curve (A) and lithium enrichment ability (B) of the strain HB04 of the present invention at different salinities.

[0022] Figure 6 It is the growth curve (A) and lithium enrichment ability (B) of the strain HB04 of the present invention at different temperatures.

[0023] Figure 7 It is the growth curve (A) and lithium enrichment ability (B) of the strain HB04 of the present invention at different pH values. Figure 8 It is the growth curve (A) and enrichment ability (B) of the strain HB04 of the present invention in produced water samples 1 and 2 of heavy oil. DETAILED DESCRIPTION OF THE INVENTION

[0024] It should be noted that the following detailed descriptions are all illustrative and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0025] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they specify the presence of features, steps, operations, devices, components, and / or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments and not for limiting the protection scope of the present invention.

[0026] In a typical specific embodiment of the present invention, a strain of Microbacterium is provided, named Microbacterium paraoxydans HB04. This strain was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on February 17, 2025, and the deposit number is CCTCC NO: M2025230.

[0027] The above-mentioned Microbacterium was isolated from Antarctic sea ice and is an aerobic Gram-positive bacterium. The colonies are rod-shaped, light yellow, opaque, full in shape, smooth and moist, easy to pick up, and the bacterial lawn grows along the streak. After 16S rDNA sequencing, it was determined to be of the genus Microbacterium and named Microbacterium paraoxydans HB04.

[0028] In another specific embodiment of the present invention, a microbial inoculant is provided, which contains the Microbacterium or its ferment or its metabolite described in the first aspect above.

[0029] The term "ferment" is used to refer to the fermentation product. The corresponding ferment can be a liquid obtained from the process of fermenting and culturing Microbacterium HB04 bacteria. Therefore, it can also be called fermentation broth; the liquid may contain bacteria (cell bodies), but it does not necessarily need to contain bacteria. The liquid preferably contains metabolites produced by the strain HB04 of the present invention.

[0030] In the present invention, the metabolite contains extracellular polymers of the strain. The Microbacterium of the present invention can specifically adsorb and enrich lithium from various other ions in produced water by its metabolic activities to produce abundant extracellular polymers.

[0031] Also, in an embodiment of the present invention, the fermentation broth or culture solution containing bacterial cells is separated from the liquid in which the bacterial cells grow in the fermentation broth or culture solution by centrifugation, filtration, sedimentation or other means known in the art, and the liquid remaining after removing the bacterial cells is the "supernatant". And in the present invention, the extracellular metabolites of strain HB04 are contained in the supernatant. In an embodiment of the present invention, the microbial inoculant may also contain the supernatant.

[0032] Also, in an embodiment of the present invention, the fermentation broth or culture solution containing bacterial cells is separated from the liquid in which the bacterial cells grow in the fermentation broth or culture solution by centrifugation, filtration, sedimentation or other means known in the art to obtain bacterial cells, and the bacterial cells can be disrupted to obtain disrupted bacterial cell products. The disruption method can be ultrasound (such as ice bath ultrasonic cell disruption) or other means known in the art. Or, further, the supernatant is collected by centrifuging the disrupted bacterial cell products, and this supernatant is denoted as cell-free extract. And in the present invention, the intracellular metabolites of strain HB04 are contained in the disrupted bacterial cell products or the cell-free extract. In an embodiment of the present invention, the microbial inoculant may also contain the disrupted bacterial cell products or the cell-free extract.

[0033] Also, in an embodiment of the present invention, the preparation method of the microbial inoculant is as follows: Inoculate strain HB04 into LB medium and culture it to obtain the product.

[0034] In the microbial inoculant, in addition to the active ingredient, a carrier is also contained. The carrier can be a carrier commonly used in the field of inoculants and is biologically inert.

[0035] The carrier can be a solid carrier or a liquid carrier;

[0036] The solid carrier can be a mineral material, a plant material or a polymer compound; the mineral material can be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material can be at least one of corn flour, bean flour and starch; the polymer compound can be polyvinyl alcohol or / and polyglycol.

[0037] The liquid carrier can be an organic solvent, a vegetable oil, a mineral oil or water; the organic solvent can be decane or / and dodecane.

[0038] The dosage form of the microbial inoculant can be various dosage forms, such as a liquid agent, an emulsion, a suspension, a powder, a granule, a wettable powder or a water dispersible granule.

[0039] According to needs, adhesives, stabilizers, etc. can also be added to the microbial inoculant, which are not specifically defined herein.

[0040] In a specific embodiment of the present invention, there is provided an application of the above-mentioned Microbacterium or the above-mentioned microbial agent as a lithium enrichment agent.

[0041] In a specific embodiment of the present invention, there is provided an application of the above-mentioned Microbacterium or the above-mentioned microbial agent in the selective enrichment and separation of lithium in a lithium-containing environment.

[0042] More specifically, in the present invention, Microbacterium can grow and reproduce at 10 - 50 °C, pH 4.0 - 11.0, and salinity 0 - 7%, and can specifically enrich lithium to a certain extent. Therefore, it can adapt to a variety of harsh lithium-containing environments, such as high-temperature or low-temperature environments, acidic or alkaline environments, and low-salt or high-salt environments.

[0043] In a specific embodiment of the present invention, in the said application, the lithium-containing environment can be a lithium-containing water environment with high temperature or low temperature, acidic or alkaline, and low salt or high salt; further, the water environment can be a (low-quality) brine environment, and further can be oilfield produced water, especially heavy oil produced water. Specific limitations are not made here.

[0044] In a specific embodiment of the present invention, there is provided a method for enriching and separating lithium from low-quality lithium-containing brine, the method comprising: applying the above-mentioned Microbacterium or the above-mentioned agent to the low-quality lithium-containing brine.

[0045] Specifically, the low-quality lithium-containing brine can be oilfield produced water, especially heavy oil produced water.

[0046] The following further illustrates the present invention with specific examples. The following examples are only for explaining the present invention and do not limit its content. Any simple modifications, equivalent changes and modifications made to the embodiments according to the technical essence of the present invention all fall within the scope of the technical solution of the present invention.

[0047] The components of each culture medium used in the following examples are as follows:

[0048] LB liquid medium: 10 g / L tryptone, 5 g / L yeast extract, 30 g / L sodium chloride.

[0049] LB solid medium: 10 g / L tryptone, 5 g / L yeast extract, 30 g / L sodium chloride, 18 g / L agar.

[0050] Example 1: Isolation, preservation, activation and identification of strain HB04

[0051] (1) Isolation of strain HB04

[0052] Antarctic sea ice was collected and several strains of bacteria were isolated. 10 mg / L of lithium chloride was added to the LB liquid medium, and 14 strains with good growth and the ability to enrich lithium were preliminarily screened out; further optimization was carried out to obtain the strain HB04 with the strongest Li + cell accumulation ability and the highest enrichment rate (shake flask experiment conditions: 10 °C, 200 r / min).

[0053] (2) Activation and preservation of strain HB04

[0054] Take 100 μL of the preserved HB04 bacterial liquid and activate it in 50 mL of LB liquid medium for 24 h to obtain a bacterial liquid with an OD 600 = 0.5 (measured after diluting the bacterial liquid 10 times).

[0055] Adopt the glycerol cryopreservation method: Transfer 0.5 mL of the activated bacterial liquid in LB liquid culture into 1 mL of 30% sterilized glycerol and store it at -80 °C.

[0056] (3) Identification of strain HB04

[0057] The colonies formed by strain HB04 are round, moist, smooth, and yellow, growing neatly along the streak. The colony plate culture photo of strain HB04 is as Figure 1 shown.

[0058] The physiological and biochemical characteristics of strain HB04 are: aerobic, Gram-positive staining, rod-shaped bacteria, some arranged at a certain angle, in a V-shape in pairs. The optical microscope picture after staining is as Figure 2 shown.

[0059] Inoculate the activated strain HB04 in LB liquid medium and shake culture at 25 °C and 200 r for 24 h. Take 1 mL of the fresh cultured bacterial liquid, centrifuge it at 4 °C and 8000 r for 5 min, collect the bacterial cells in a 2 mL centrifuge tube, and extract DNA using a DNA extraction kit. After electrophoresis detection, PCR amplification is carried out using the 16S rDNA gene universal primers 7F / 1540R respectively. After electrophoresis detection of the PCR products, the 16S rDNA gene sequence is determined, and the specific sequence of the 16S rDNA gene is as shown in SEQ D NO.1.

[0060] By uploading the 16S rDNA sequence of strain HB04 to the National Center for Biotechnology Information website, comparing it with the standard strains on the website, and using the MEGA-X software to construct a bacterial phylogenetic tree as Figure 3, thus determining that the strain HB04 belongs to the genus Microbacterium and is named Microbacterium paraoxydans HB04. This strain was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on February 17, 2025, and the deposit number is CCTCC NO: M 2025230.

[0061] For the convenience of description, Microbacterium paraoxydans HB04 described in the present invention will also be abbreviated as strain HB04 or HB04 in the application documents.

[0062] Example 2: Initial Li + Effect of concentration on the growth and lithium enrichment of strain HB04

[0063] Take the activated bacterial liquid in Example 1 and inoculate it into fresh LB liquid medium without Li + , 2mg / L Li + , 4mg / L Li + , 8mg / L Li + , 10mg / L Li + , 20mg / L Li + , 30mg / L Li + so that the initial cell concentration is 0.013 in terms of OD 600 (measured after diluting the bacterial liquid 10 times). Design 3 parallel samples and 1 blank control group without inoculating the strain. Place it in a constant temperature incubator at 25°C and a rotation speed of 200r for cultivation. Regularly take samples in the ultra-clean workbench to measure the OD value of the bacteria, and use atomic absorption spectrometry to measure the Li + enrichment rate at 48h, calculate the Li + cell accumulation amount, and the results are shown in Figure 4 .

[0064] It has been experimentally proven that Li + within the range of 0 - 30mg / L has almost no effect on the growth of strain HB04, and the OD value of the bacteria continuously increases to above 0.9 (measured after diluting the bacterial liquid 10 times). And with the increase of the Li + concentration, the Li + cell accumulation amount and Li + enrichment rate at 48h increase significantly. When Li + is 30mg / L, the Li + cell accumulation amount and Li + enrichment rate reach 8.32 mmol / g cell dry weight and 69.57% respectively.

[0065] Example 3: Effects of salinity on the growth and lithium enrichment of strain HB04

[0066] Take the activated bacterial liquid in Example 1 and inoculate it into fresh LB liquid media with salinities of 0%, 1%, 2%, 3%, 4%, 5%, 6%, and 7% respectively, so that the initial cell concentration is 0.013 in terms of OD 600 (measured after diluting the bacterial liquid 10 times). Design 3 parallel samples and 1 blank control group without inoculating the strain. Place them in a constant temperature incubator at 25°C with a rotation speed of 200 r for cultivation. Regularly take samples in a laminar flow hood to measure the OD value of the cells, and use atomic absorption spectrometry to measure the Li + enrichment rate, and calculate the Li + cellular accumulation. The results are shown in Figure 5 .

[0067] It has been experimentally proven that strain HB04 can grow within the salinity range of 0 - 7%. The OD value of the cells continuously increases to above 0.8 (salinity of 7%) to 0.9 (salinity of 3%) (measured after diluting the bacterial liquid 10 times), showing good salt tolerance. Li + enrichment can be achieved at salinities of 0 - 7%. The lithium enrichment rate can reach 69% at a salinity of 2%. The Li + accumulation in HB04 exceeds 2.74 mmol / g cell dry weight after 48 h.

[0068] Example 4: Effects of reaction temperature on the growth and lithium enrichment of strain HB04

[0069] Take the activated bacterial liquid in Example 1 and inoculate it into fresh LB liquid medium, so that the initial cell concentration is 0.013 in terms of OD 600 (measured after diluting the bacterial liquid 10 times), and place them in a constant temperature incubator at 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C with a rotation speed of 200 r for cultivation. Design 3 parallel samples and 1 blank control group without inoculating the strain. Regularly take samples in a laminar flow hood to measure the OD value of the cells, and use atomic absorption spectrometry to measure the Li + enrichment rate, and calculate the Li + cellular accumulation. The results are shown in Figure 6 .

[0070] It has been experimentally proven that strain HB04 can grow in large quantities within the temperature range of 10°C - 50°C. The OD value of the cells continuously increases to above 0.6 (50°C) to 1.0 (35°C) (measured after diluting the bacterial liquid 10 times), showing good tolerance to high and low temperatures. The Li + accumulation remains above 2.48 mmol / g cell dry weight after 48 h, and the Li + enrichment rate can reach up to 70.06%.

[0071] Example 5: Effect of Initial pH on the Growth of Strain HB04 and Lithium Enrichment

[0072] Use 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide to adjust the pH of LB liquid medium to 3, 4, 5, 6, 7, 8, 9, 10, and 11 respectively, and inoculate the activated bacterial liquid in Example 1 so that the initial cell concentration is OD 600 counted as 0.013 (measured after diluting the bacterial liquid 10 times), place it in a constant temperature incubator at 25 °C and a rotation speed of 200 r for cultivation, regularly sample in a laminar flow bench to measure the OD value of the bacteria, and use atomic absorption spectrometry to measure the Li + enrichment rate, and calculate the Li + cell accumulation. The results are shown in Figure 7 .

[0073] It has been experimentally proven that strain HB04 can grow in large quantities within the pH range of 4 - 11, has a wide adaptation range, good alkali tolerance, and the lithium enrichment ability does not show obvious attenuation within the pH range of 5 - 8. The Li + accumulation remains above 2.50 mmol / g cell dry weight in 48 h, and the Li + enrichment rate can reach up to 65.84%.

[0074] Example 6: Growth Curve and Lithium Enrichment Efficiency of Strain HB04 in Actual Heavy Oil Produced Water

[0075] Take the activated bacterial liquid in Example 1, centrifuge at 8000 r at 4 °C for 10 min to retain the bacteria, wash them repeatedly 3 times with 2.5% normal saline, and inoculate them into the sterilized heavy oil produced water samples 1 and 2 from different wellheads (lithium ion concentrations are 3.03 mg / L and 6.93 mg / L respectively) at a ratio of 10%. Make the initial cell concentration OD 600 counted as 0.04 (measured after diluting the bacterial liquid 10 times), and add additional nutrients (calculated as sodium acetate to make C / N 25), and place them in a constant temperature incubator at 25 °C and a rotation speed of 200 r for cultivation. Regularly sample in a laminar flow bench to measure the OD value of the bacteria, and use atomic absorption spectrometry to measure the Li + enrichment rate, and calculate the Li + cell accumulation. The results are shown in Figure 8 .

[0076] It has been experimentally proven that strain HB04 has the function of enriching lithium in actual heavy oil produced water. In produced water sample 1, the Li + accumulation remains above 0.57 mmol / g cell dry weight in 72 h, and the Li + enrichment rate is about 31.34%; in produced water sample 2, the Li+ The cumulative amount remains above 1.59 mmol / g of cell dry weight, Li + The enrichment rate is approximately 28.66%.

[0077] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the given examples, those of ordinary skill in the art can modify or equivalently replace the technical solution of the present invention as needed, without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A strain of Microbacterium, named Microbacterium paraoxydans HB04, which was deposited at the China Center for Type Culture Collection on February 17, 2025, with the deposit number CCTCC NO: M 2025230.

2. A microbial inoculant, characterized in that, It contains the Microbacterium described in claim 1, or its fermented product, or its metabolite.

3. The microbial inoculum according to claim 2, wherein The metabolite contains extracellular polymeric substances.

4. Use of the Microbacterium described in claim 1 or the microbial agent described in any one of claims 2 - 3 as a lithium enrichment agent.

5. Use of the Microbacterium described in claim 1 or the microbial agent described in any one of claims 2 - 3 in the selective enrichment and separation of lithium in a lithium-containing environment.

6. The application according to claim 5, wherein The lithium-containing environment is a lithium-containing water environment with high or low temperature, acidic or alkaline, and low or high salt.

7. The application according to claim 6, wherein, The water environment is a low-quality brine environment, further an oilfield produced water, and even further a viscous oil produced water.

8. The application according to claim 5, wherein The temperature of the lithium-containing environment is 10 - 50 °C, pH 4.0 - 11.0, and salinity 0 - 7%.

9. A method for enriching and separating lithium from low-quality brine containing lithium, characterized in that, The method includes: applying the Microbacterium described in claim 1 or the microbial agent described in any one of claims 2 - 3 to the lithium-containing low-quality brine.

10. The method according to claim 9, characterized in that, The lithium-containing low-quality brine is an oilfield produced water, further a viscous oil produced water.

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