Pseudomonas kunmingensis and uses thereof

Abstract

The present application relates to the field of environment-friendly biological exploitation of mineral resources, and particularly relates to Pseudomonas kunmingensis and application thereof.The present application provides Pseudomonas kunmingensis TR-21, and the preservation number of which is CGMCC No.26447.The present application also provides a recombinant strain.The strain provided by the present application has low culture cost, fast growth speed, simple leaching conditions, and the ability to leach various rare earth ions, and has a good application prospect in the aspects of rare earth microbial leaching and tailing utilization.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of environment-friendly biological exploitation of mineral resources, in particular to Pseudomonas kunmingensis and its application. BACKGROUND

[0002] Rare earth elements include fifteen lanthanide elements and two metallic elements of scandium and yttrium, which are widely used in cutting-edge research and high-tech industries as an important strategic resource, and are known as "industrial gold". China is the country with the most abundant rare earth resources in the world, with complete rare earth minerals and elements, reasonable mineral grade and resource distribution. However, the illegal mining in the early years and the low utilization development mode have led to a sharp decrease in the reserves of rare earth resources in China. However, the existing rare earth ore processing methods, such as sulfuric acid roasting method, alkali method and ammonium salt leaching method, will cause environmental pollution to some extent, and the subsequent solvent extraction also mostly uses organic solvents such as organic acids and phosphoric acid esters, which will also cause different degrees of environmental pollution. Therefore, it is urgent to develop a clean and efficient new leaching technology to ensure the leaching rate of rare earth elements while minimizing the pollution to the environment.

[0003] The method of leaching valuable metal elements by the direct action of microorganisms and the action of metabolic products is considered as an important technology of clean and efficient leaching because of its green environmental protection, simple operation and low cost. For example, Acidithiobacillus ferrooxidans can utilize the oxidation-reduction reaction between iron and sulfur-containing minerals to provide energy for growth under acidic conditions, thereby completing the leaching of copper. However, there are few reports on the biological leaching of rare earth elements in tailings. SUMMARY

[0004] Therefore, the present application provides Pseudomonas kunmingensis and its application. The present application provides Pseudomonas kunmingensis TR-21, which has a preservation number of CGMCC No. 26447 and a recombinant strain. The present application finds through experiments that the strain provided by the present application has low culture cost, fast growth speed, simple leaching conditions, and leaching capacity for various rare earth ions, and has good application prospect in rare earth microbial leaching and tailings utilization.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides Pseudomonas kunmingensis TR-21, which has a preservation number of CGMCC No. 26447.

[0007] In some embodiments of the present application, the Pseudomonas kunmingensis TR-21 includes one or more of live bacteria, inactivated bacteria, fermentation broth, exosome or metabolite thereof.

[0008] The present application also provides a method for preparing a recombinant strain based on the Pseudomonas kunmingensis TR-21, wherein the recombinant strain is obtained by any of the following:

[0009] (I) knocking out or knocking down the coding gene PhoX of alkaline phosphatase; and / or

[0010] (II) overexpressing the coding gene gltA of citrate synthase.

[0011] In some embodiments of the present application, the method for preparing a recombinant strain includes the following steps:

[0012] Step 1, inserting a target gene into a vector plasmid to construct a recombinant plasmid;

[0013] Step 2, transforming the recombinant plasmid into TR-21 to express and obtain a recombinant strain.

[0014] In some embodiments of the present application, the target gene includes knocking out or knocking down the coding gene PhoX of alkaline phosphatase; and / or

[0015] overexpressing the coding gene gltA of citrate synthase.

[0016] In some embodiments of the present application, the recombinant plasmid further includes a 6×His tag.

[0017] In some embodiments of the present application, the vector plasmid includes pCM62 or pCM66.

[0018] The present application also provides a recombinant strain obtained by the method.

[0019] The present application also provides a microbial inoculant, which includes any of the following:

[0020] (I) the Pseudomonas kunmingensis TR-21; and / or

[0021] (II) the recombinant strain.

[0022] The present application also provides an application of any of the following in the utilization of tailings:

[0023] (I) the Pseudomonas kunmingensis TR-21; and / or

[0024] (II) the recombinant strain; and / or

[0025] (III) the microbial inoculant.

[0026] The present application also provides the use of any of the following in the bioleaching of rare earth elements:

[0027] (I) the Pseudomonas kunmingensis TR-21; and / or

[0028] (II) the recombinant strain; and / or

[0029] (III) the microbial inoculant.

[0030] In some embodiments of the present application, the rare earth elements comprise rare earth elements in tailings.

[0031] In some embodiments of the present application, the rare earth elements comprise La, Ce, Pr, Nd, MREE or HREE.

[0032] The present application also provides a method of producing rare earth elements, comprising producing rare earth elements based on any of the following:

[0033] (I) the Pseudomonas kunmingensis TR-21; and / or

[0034] (II) the recombinant strain; and / or

[0035] (III) the microbial inoculant.

[0036] In some embodiments of the present application, the method of producing comprises mixing, culturing, filtering, and producing rare earth elements from any of the following:

[0037] (I) the Pseudomonas kunmingensis TR-21; and / or

[0038] (II) the recombinant strain; and / or

[0039] (III) the microbial inoculant.

[0040] In some embodiments of the present application, the method of producing comprises:

[0041] the raw material comprises tailings; and / or

[0042] The culture temperature comprises 30 DEG C.

[0043] In some embodiments of the present application, the method for preparing rare earth elements comprises the following steps:

[0044] Step 1, culture of microorganism: Pseudomonas kunmingensis TR-21 is cultured in Luria-Bertani (LB) liquid medium at 30 DEG C and 200 rpm overnight.

[0045] Step 2, preparation of microbial inoculum: the bacterial suspension in step 1 is centrifuged at 8000 rpm for 5 min to collect the bacterial precipitate, which is washed with ultrapure water for 3 times.

[0046] Step 3, leaching of rare earth elements: the bacterial precipitate in step 2 is resuspended with ultrapure water, mixed with rare earth tailings in a certain proportion, and leached at 30 DEG C and 200 rpm, and the leaching solution is obtained after 5 days to prepare rare earth elements.

[0047] The present application has the following beneficial effects, including but not limited to:

[0048] The present application provides a method for leaching rare earth elements from tailings by using microorganisms and their metabolites, which solves the problems of large amount of chemical reagents, high production cost and serious environmental pollution in the process of chemical leaching of rare earth tailings.

[0049] The strain provided by the present application has low culture cost, fast growth speed, simple leaching conditions, and leaching ability for various rare earth ions, and has good application prospect in rare earth microbial leaching and tailings utilization.

[0050] Biological preservation instructions

[0051] Biological material: TR-21, classification and naming: Pseudomonas kunmingensis, preserved in the China General Microbiological Culture Collection Center on January 12, 2023, address: No. 3, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology of Chinese Academy of Sciences, preservation number: CGMCC No. 26447.

[0052] The TR-21 in the present application is the strain with the above preservation number CGMCC No. 26447. BRIEF DESCRIPTION OF DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or the prior art description will be briefly introduced as follows.

[0054] Figure 1 Figure showing the map of recombinant plasmid pCM62 (carrying alkaline phosphatase-PhoX);

[0055] Figure 2 Figure showing the map of recombinant plasmid pCM62 (carrying citrate synthase-gltA);

[0056] Figure 3 Figure showing SDS-PAGE detection of alkaline phosphatase expressed by TR-21;

[0057] Figure 4 Figure showing SDS-PAGE detection of citrate synthase expressed by TR-21. DETAILED DESCRIPTION

[0058] The present application discloses Pseudomonas kunmingensis and its application, and those skilled in the art can refer to the content herein to realize the process parameters by appropriate improvement. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are regarded as included in the present application. The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0059] The present application provides a method for leaching rare earth elements in tailings by microorganisms and their metabolites, which solves the problems of large amount of chemical reagents, high production cost and serious environmental pollution in the process of chemical leaching of rare earth tailings.

[0060] To achieve the above object, the present application adopts the following technical solutions:

[0061] (1) Screening of leaching strain: the strain is screened by using phosphorus solubilizing medium Pikovskaya (PK) solid medium. Pseudomonas kunmingensis TR-21 which can produce obvious phosphorus solubilizing transparent ring on PK solid medium is selected for subsequent research.

[0062] (2) Culture of microorganism: Pseudomonas kunmingensis TR-21 is cultured in Luria-Bertani (LB) liquid medium at 30℃ and 200rpm overnight.

[0063] (3) Preparation of microbial inoculant: the bacterial suspension in step (1) is centrifuged at 8000rpm for 5min to collect bacterial precipitate, which is washed with ultrapure water for 3 times.

[0064] (4) Leaching rare earth elements: the bacteria in step (3) are resuspended with ultrapure water, mixed with rare earth tailings in proportion, and placed at 30℃, 200rpm for leaching. After 5 days, the leaching solution is obtained.

[0065] (5) The content and type of rare earth elements in step (4) are detected by ICP-MS to identify the leaching ability of TR-21.

[0066] (6) Genetic engineering technology is used to modify the TR-21 strain: a plasmid is used as an expression vector, a target gene fragment is inserted to obtain a recombinant plasmid, and the recombinant plasmid is electroporated into TR-21. By expressing the target gene in TR-21, the expression level of intracellular phosphatase and organic acid synthesis pathway is up-regulated.

[0067] Therefore, the present application provides a new Pseudomonas kunmingensis strain capable of leaching rare earth ions from tailings, which can provide new options and approaches for bioleaching. The strain provided by the present application has low culture cost, fast growth rate, simple leaching conditions, and leaching ability for various rare earth ions, and has good application prospect in rare earth microbial leaching and tailings utilization.

[0068] Unless otherwise specified, the raw materials and reagents used in the Pseudomonas kunmingensis and its application provided by the present application can be purchased from the market.

[0069] The present application will be further described below in conjunction with examples:

[0070] Example 1: Screening of leaching bacteria and determination of the ability to dissolve insoluble phosphate

[0071] All strains that need to be verified for phosphorus solubilizing ability are picked from the solid culture medium to 4mL LB liquid medium after purification, and incubated at 30℃, 200rpm in a constant temperature shaking incubator overnight. 2μL of bacterial solution is added to Pikovskaya solid medium, and after ten days of culture, the results are observed and recorded. The presence of a transparent ring is recorded as positive, indicating that the strain has the ability to dissolve insoluble phosphate. It is found that the residual LB medium has a negative effect on the production of transparent ring by bacteria on Pikovskaya solid medium. Because of the residual nutrients (such as peptone and yeast extract) in LB medium, the strain grows too fast, affecting the observation of transparent ring. Therefore, in the subsequent experiment, the bacterial solution after overnight culture is washed with Milli-Q ultrapure water for 3 times, and then 2μL of the bacterial solution resuspended with ultrapure water is added to Pikovskaya solid medium to observe whether a transparent ring is produced. It is found that Pseudomonas kunmingensis (CGMCC NO.: 26447) TR-21 produces a transparent ring on Pikovskaya solid medium.

[0072] Preparation of TR-21 microbial inoculant

[0073] The purified TR-21 single colony was picked from the solid culture medium into 4 mL of LB liquid medium and incubated overnight at 30°C with 200 rpm constant temperature shaking in a shaking incubator as seed liquid. 1% of the seed liquid was inoculated into LB liquid medium and incubated at 30°C with 200 rpm constant temperature shaking until the logarithmic phase. The bacterial cells were collected by centrifugation at 8000 rpm for 5 min, washed with sterile ultrapure water for 3 times, weighed, and TR-21 microbial inoculant was prepared.

[0074] Example 3: Detection of the ore leaching capacity of TR-21 by ICP-MS

[0075] The bacterial cells (wet weight 1.0 g) washed and weighed in Example 2 were added to 100 mL of sterile ultrapure water, and the same weight of sterile tailings as the bacterial cell precipitate was added. The ore leaching was carried out in a constant temperature shaking incubator at 30°C with 200 rpm for 7 days.

[0076] 5 mL of the leached culture liquid was taken at 1 d, 2 d, 3 d, 4 d, 5 d, 6 d and 7 d, respectively, and centrifuged at 8000 rpm for 5 min. The supernatant was taken and filtered with a 0.22 μm needle filter. The filtrate was collected. The rare earth elements in the filtrate were qualitatively and quantitatively analyzed by ICP-MS, and the results are shown in Table 1. The content of rare earth elements in the solution continued to increase during the first 4 days of ore leaching.

[0077] Table 1: Leaching amount of rare earth elements in tailings by TR-21 strain (μg / L)

[0078]

[0079]

[0080] Example 4: Modification of TR-21 strain by genetic engineering technology

[0081] (1) Inserting the target gene fragment into the vector plasmid

[0082] The target gene sequences, such as PhoX gene (PP4_RS21520, ID: 45525756) encoding alkaline phosphatase (76.67 kDa in size) and gltA gene (ID: 45670758) encoding citrate synthase (47.85 kDa in size) involved in the synthesis of citrate metabolic pathway, were obtained from the DNA sequence database (GenBank, National Center for Biotechnology Information, USA). The vector plasmid pCM62 and the target gene were cleaved by the same restriction enzyme, such as type II restriction enzyme HindIII and XbaI, to generate the same sticky ends. After adding an appropriate amount of DNA ligase, the target gene fragment was inserted into the cut vector plasmid to form a recombinant plasmid with the target gene (e.g. Figure 1 and Figure 2 ). Meanwhile, a 6×His tag gene was inserted into the plasmid to facilitate detection of whether the target gene can be expressed in TR-21 cells.

[0083] (2) Transformation of the recombinant plasmid into TR-21 for expression

[0084] The TR-21 strain was treated with CaCl2to form competent cells. The recombinant vector plasmid was incubated with the competent cells, and the target gene and marker gene were transformed into the cells along with the plasmid. SDS-PAGE was used to detect whether the target gene was expressed, and the results are shown in Figure 3 and Figure 4 , which indicates that the target gene has been successfully introduced into the TR-21 cells and can be maintained stably and expressed in TR-21, obtaining an engineered TR-21 strain (recombinant strain). The leaching capacity of the engineered TR-21 strain was detected by the methods of Example 2 and Example 3. The results are shown in Table 2, and the introduction of the recombinant plasmid pCM62 carrying citrate synthase-gltA significantly improves the leaching capacity of the TR-21 strain (P<0.05), while the introduction of the recombinant plasmid pCM62 carrying alkaline phosphatase-PhoX not only fails to improve the leaching capacity, but also significantly reduces the leaching capacity of TR-21 (P<0.05).

[0085] Table 2 Leaching amount of rare earth elements in tailings by engineered TR-21 strain (μg / L)

[0086]

[0087] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. *Pseudomonas kunmingense* ( Pseudomonas kunmingensis TR-21, characterized in that, The preservation number thereof is CGMCC No. 26447.

2. The *Pseudomonas kunmingense* as described in claim 1 (… Pseudomonas kunmingensis Fermentation broth of TR-21.

3. A method for preparing a recombinant strain, characterized by, The recombinant strain of Pseudomonas kumeongensis according to claim 1, Pseudomonas kunmingensis ) TR-21 or the fermentation broth according to claim 2, said recombinant strain is obtained by overexpression of the coding gene gltA for citrate synthase.

4. The recombinant strain obtained by the preparation method of claim 3.

5. A microbial inoculant characterized in that, It comprises any of the following: (I) Pseudomonas kumlungiensis (Pku) according to claim 1 Pseudomonas kunmingensis ) TR-21 ; and / or (II), the fermentation liquor of claim 2; and / or (III), the recombinant strain of claim 4.

6. Application of any of the following in tailings utilization: (I) Pseudomonas kumlungiensis (Pku) according to claim 1 Pseudomonas kunmingensis ) TR-21 ; and / or (II), the fermentation liquor of claim 2; and / or (III), the recombinant strain of claim 4; and / or (IV), the microbial inoculant of claim 5; the tailings utilization is leaching of rare earth elements from rare earth tailings; The rare earth elements are selected from La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho or Er.

7. Application of any of the following in rare earth element bioleaching: (I) Pseudomonas kumlungiensis (Pku) according to claim 1 Pseudomonas kunmingensis ) TR-21 ; and / or (II), the fermentation liquor of claim 2; and / or (III), the recombinant strain of claim 4; and / or (IV), the microbial inoculant of claim 5; The rare earth element bioleaching refers to leaching of rare earth elements from rare earth tailings; The rare earth elements are selected from La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho or Er.

8. A method for producing a rare earth element, characterized by, Leaching of rare earth elements from rare earth tailings based on any of the following: (I) Pseudomonas kumlungiensis (Pku) according to claim 1 Pseudomonas kunmingensis ) TR-21 ; and / or (II), the fermentation liquor of claim 2; and / or (III), the recombinant strain of claim 4; and / or (IV), the microbial inoculant of claim 5; The rare earth elements are selected from La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho or Er.

9. The production method according to claim 8, wherein It comprises mixing any of the following with raw materials, culturing, filtering, and preparing rare earth elements: (I) Pseudomonas kumlungiensis (Pku) according to claim 1 Pseudomonas kunmingensis ) TR-21 ; and / or (II), the fermentation liquor of claim 2; and / or (III), the recombinant strain of claim 4; and / or (IV), the microbial inoculant of claim 5.

10. The production method according to claim 9, wherein It comprises: The culturing temperature comprises 30℃.

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