Method for synthesizing red elemental selenium by citrobacter sp. and biological application thereof

Abstract

The present application relates to the field of microbial technology, and particularly relates to a method for synthesizing red elemental selenium by using phosphorus-dissolving arsenate citrobacter and biological application. The present application aims to solve the technical problem of how to prepare bioactive red elemental selenium from a strain of bacteria which grows rapidly and has the function of dissolving organic phosphorus, and apply the obtained elemental selenium to nitrogen-fixing rhizobium to improve its stress resistance and promote its growth. To this end, the present application provides a strain of arsenate citrobacter (Citrobacter arsenatis), which has a preservation number of CGMCC No. 33109, provides a method for preparing red elemental selenium by using the strain, and applies the red elemental selenium to nitrogen-fixing rhizobium. The present application has the advantages that the strain can efficiently degrade organic phosphorus, grow rapidly, and synthesize red elemental selenium; and the application of the red elemental selenium to rhizobium improves the drought resistance and growth performance of the rhizobium.

Description

Technical Field

[0001] The present application belongs to the field of microbial technology, and specifically relates to a method for synthesizing red elemental selenium by phosphate-arsenate-soluble Citrobacter and its biological application. Background Art

[0002] Phosphorus is the second most essential element for plant growth. However, soil phosphorus is often fixed or trapped in biomacromolecules, making it difficult for plants to directly absorb and utilize it, thus limiting plant growth. Phosphorus-solubilizing bacteria are considered important plant growth-promoting bacteria because they can release poorly soluble phosphorus from soil or biomacromolecules for their own absorption and utilization by themselves and plants. These bacteria play a vital role in increasing soil phosphorus availability and promoting plant growth.

[0003] Selenium is an essential trace element, and appropriate amounts of it have a positive effect on plant growth and stress resistance. However, when the concentration of sodium selenite solution containing selenium is too high, it can be toxic to plants. Fortunately, certain microorganisms are able to convert sodium selenite into non-toxic red elemental selenium. This conversion process not only reduces the toxicity of high-concentration sodium selenite to plants, but the resulting red elemental selenium can also help plants enhance their stress resistance. Therefore, this microbial conversion technology has been widely used in agricultural production.

[0004] Different microorganisms have different abilities to solubilize poorly soluble phosphorus and reduce sodium selenite to red elemental selenium, and the time required also varies. Therefore, it is urgent to identify more efficient phosphate-solubilizing bacteria and strains that can quickly convert sodium selenite to red elemental selenium.

[0005] Rhizobia are important beneficial microorganisms in agricultural production, playing a key role in promoting nodulation and nitrogen fixation in legumes, improving the yield and quality of legume crops, reducing the use of chemical nitrogen fertilizers, and protecting the ecological environment. However, the application of rhizobia to legume seeds or soil often faces the challenge of drought stress. Improving the drought tolerance of rhizobia and ensuring their vitality in adverse environments is a prerequisite for fully realizing their symbiotic nitrogen-fixing function.

[0006] In summary, the development of efficient microbial resources, such as phosphate-solubilizing bacteria and sodium selenite-reducing bacteria, combined with the application of rhizobia, is expected to provide innovative solutions for improving the sustainability and stress resistance of agricultural production. Summary of the Invention

[0007] The technical problems to be solved by the present invention are how to obtain a bacterium capable of both solubilizing organic phosphorus and synthesizing red elemental selenium, how to prepare elemental selenium, and how to effectively utilize the prepared elemental selenium. The technical problems to be solved are not limited to the technical subject matter described herein, and those skilled in the art will clearly understand other technical subjects not described herein through the following description.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The invention provides a Citrobacter arsenatis strain, the original strain number of which is GY-C-Y7, and the registration and preservation number of which in the General Microbiology Center of China Culture Collection of Microorganisms is CGMCC No.33109.

[0010] Those skilled in the art will appreciate that variants or descendants of the aforementioned Citrobacter arsenate CGMCC No. 33109 also fall within the scope of protection of the present invention. The variants or descendants of the aforementioned Citrobacter arsenate CGMCC No. 33109 have similar or identical functions to the aforementioned Citrobacter arsenate CGMCC No. 33109.

[0011] In certain embodiments, the variant or progeny of Citrobacter arsenate CGMCC NO.33109 has similar or identical physiological and biochemical properties to that of Citrobacter arsenate CGMCC NO.33109.

[0012] In certain embodiments, the Citrobacter arsenicum CGMCC NO.33109 has one or more functions selected from the following:

[0013] Q1) dissolving organic phosphorus;

[0014] Q3) reducing sodium selenite to red elemental selenium;

[0015] Q3) Promote the growth of rhizobia under drought stress conditions.

[0016] The present invention also provides a composition containing the aforementioned Citrobacter arsenate.

[0017] The composition mentioned above may be a culture, wherein the culture is a substance obtained by culturing the Citrobacter arsenate in a microbial culture medium.

[0018] The substance is the content in the culture container.

[0019] The composition has at least one of the following properties or uses:

[0020] U1) dissolving organic phosphorus or preparing products for dissolving organic phosphorus;

[0021] U2) reducing sodium selenite to red elemental selenium or using it to prepare a product in which selenite is reduced to red elemental selenium;

[0022] U3) for preparing a product for promoting the growth of rhizobia under drought stress conditions.

[0023] The above-mentioned culture can be a substance obtained by culturing the Citrobacter arsenate in a microbial culture medium (i.e., a fermentation product, such as a fermentation broth containing the Citrobacter arsenate and a substance secreted into a liquid culture medium, or a solid fermentation product containing the Citrobacter arsenate and a substance secreted into a solid culture medium).

[0024] The composition mentioned above can be a bacterial agent, a phosphate-dissolving preparation, or a preparation for preparing red elemental selenium.

[0025] In certain embodiments, the Citrobacter arsenate may be in a solid form, such as a dried or freeze-dried culture preparation.

[0026] The above composition has at least one of the following uses:

[0027] A1) Dissolved organic phosphorus;

[0028] A2) preparing a product for dissolving organophosphorus;

[0029] A3) reducing sodium selenite to red elemental selenium;

[0030] A4) preparing a product by reducing selenite to red elemental selenium;

[0031] A5) Promote the growth of rhizobia under drought stress conditions;

[0032] A6) Preparation of a product that promotes the growth of rhizobia under conditions of drought stress.

[0033] The present invention also provides an application of Citrobacter arsenate, wherein the application is any one of the following:

[0034] B1) Dissolved organic phosphorus;

[0035] B2) preparing a product for dissolving organophosphorus;

[0036] B3) reducing sodium selenite to red elemental selenium;

[0037] B4) preparing a product by reducing selenite to red elemental selenium;

[0038] B5) Promote the growth of rhizobia under drought stress conditions;

[0039] B6) Preparation of a product that promotes the growth of rhizobia under drought stress conditions.

[0040] The present invention also provides an application of the aforementioned composition, wherein the application is any of the following:

[0041] C1) dissolving organic phosphorus;

[0042] C2) preparing a product for dissolving organophosphorus;

[0043] C3) reducing sodium selenite to red elemental selenium;

[0044] C4) preparing a product of reducing selenite to red elemental selenium;

[0045] C5) promotes the growth of rhizobia under drought stress conditions;

[0046] C6) Preparation of a product that promotes the growth of rhizobia under conditions of drought stress.

[0047] In the above-mentioned use, the selenite may be a soluble selenite, such as sodium selenite.

[0048] The present invention also provides a method for preparing red elemental selenium, which comprises culturing the aforementioned Citrobacter arsenate in a culture medium containing selenite, collecting the bacterial cells, and extracting and purifying the red elemental selenium from the bacterial cells.

[0049] The present invention also provides a method for culturing Citrobacter arsenate, wherein the Citrobacter arsenate is a strain numbered CGMCC No. 33109. The method comprises the step of culturing the Citrobacter arsenate in a culture medium for culturing microorganisms.

[0050] In certain embodiments, the culture medium for culturing a microorganism comprises a solid or liquid culture medium, or a component of the culture medium.

[0051] In certain embodiments, the culture medium may specifically be LB liquid culture medium.

[0052] The present invention also provides a method for preparing a composition. The composition is the aforementioned composition, and the method comprises the step of using the aforementioned Citrobacter arsenate as a component of the composition.

[0053] A method for preparing a fermentation product comprises culturing the aforementioned Citrobacter arsenate to obtain a fermentation product, wherein the fermentation product has a phosphorus-dissolving function.

[0054] The term "fermentation product" encompasses all materials within a culture vessel and refers to any liquid or solid product containing a microbial population after artificial inoculation and culture. This refers to a product obtained by growing and / or amplifying microorganisms. It can be a biologically pure culture of the microorganism or contain a certain amount of culture medium, metabolites, and / or other components produced during the culture process. The term "fermentation product" also includes subcultures obtained by subculturing microorganisms, which can be cultures of a single generation or a mixture of several generations.

[0055] Herein, the metabolite can be obtained from the fermentation broth of Citrobacter arsenate CGMCC NO.33109. The metabolite of Citrobacter arsenate CGMCC NO.33109 can be a sterile metabolite of Citrobacter arsenate CGMCC NO.33109 or a bacterial metabolite of Citrobacter arsenate CGMCC NO.33109. The sterile metabolite of Citrobacter arsenate CGMCC NO.33109 (sterile fermentation filtrate) can be prepared according to the following method: Citrobacter arsenate CGMCC NO.33109 is cultured in a liquid culture medium, and Citrobacter arsenate CGMCC NO.33109 is filtered out of the liquid culture (fermentation broth) to obtain the sterile metabolite of Citrobacter arsenate CGMCC NO.33109. The bacterial metabolites of Citrobacter arsenate CGMCC NO.33109 can be prepared according to the following method: Citrobacter arsenate CGMCC NO.33109 is cultured in a liquid fermentation medium, and the fermentation broth (containing Citrobacter arsenate CGMCC NO.33109 and substances secreted into the liquid culture medium) is collected. The fermentation broth is the bacterial metabolites of Citrobacter arsenate CG MCC NO.33109.

[0056] The present invention also provides a method for using red elemental selenium synthesized by Citrobacter acidobacter in a culture of rhizobia, thereby helping the rhizobia to improve its ability to tolerate drought stress.

[0057] The present invention provides a dual-functional strain of Citrobacter arsenicum. This strain not only dissolves difficult-to-degrade lecithin, releasing the phosphorus present, but also rapidly reduces and converts colorless sodium selenite into red elemental selenium. Furthermore, the red elemental selenium synthesized by this strain can be widely used in the cultivation of rhizobia. It not only enhances the tolerance of rhizobia to drought conditions but also promotes their proliferation.

[0058] Preservation Instructions

[0059] Chinese name of the strain: Citrobacter arsenate

[0060] Latin name: Citrobacter arsenatis

[0061] Classification name: Citrobacter arsenatis

[0062] Strain ID: GY-C-Y7

[0063] Depository: General Microbiology Center of China Culture Collection Administration

[0064] Abbreviation of depository unit: CGMCC

[0065] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0066] Deposit date: December 18, 2024

[0067] The registration number of the CGMCC Collection Center is: CGMCC No.33109. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 This is a phylogenetic tree of representative species of the genus Citrobacter constructed based on 16S rDNA sequences. The reference strains used include: Citrobacter arsenatis LY-1 T ), Citrobacter europaeus 97 / 79 T ), Citrobacter freundii ATCC 8090 T ), Citrobacter youngae (C. youngae CIP 105016 T ), Citrobacter gillenii (C.gillenii CIP 106783 T sedlakii CIP 105037 T ), C.bitternis SKKU-TP7 T ), the outgroup reference strain used was: Xenorhabdus nematophila DSM 3370 T ).

[0069] Figure 2 This is a phylogenetic tree of representative species of the genus Citrobacter constructed based on the housekeeping gene recN sequence. The reference strains used include: Citrobacter arsenatis LY-1 T ), Citrobacter europaeus 97 / 79 T ), Citrobacter freundii ATCC 8090 T ), Citrobacter youngae (C. youngae CIP 105016 T ), Citrobacter gillenii (C.gillenii CIP 106783 T sedlakii CIP 105037 T), C.bitternis SKKU-TP7 T ), the outgroup reference strain used was: Xenorhabdus nematophila DSM 3370 T ).

[0070] Figure 3 The image on the left shows the results of the CGMCC No. 33109 strain after 3 days of culture, and the image on the right shows the results after 8 days of culture.

[0071] Figure 4 It is solid red elemental selenium produced by the CGMCC No.33109 strain and stored in a centrifuge tube.

[0072] Figure 5a The above figure shows the OD of rhizobium CCBAU 33470 after culturing in TY liquid culture medium for 48 hours. 600nm Results. CK is the control, indicating no addition of elemental selenium and sodium selenite; Se 0 =Indicates the addition of elemental selenium, Se indicates the addition of sodium selenite; the amounts of selenium were 5, 10, 20, and 40 μM, respectively. “*” indicates p < 0.05; “**” indicates p < 0.01; and “***” indicates p < 0.001.

[0073] Figure 5b is the OD value of rhizobium CCBAU 33470 after culturing in TY liquid culture medium for 72 hours 600nm Results. CK is the control, indicating no addition of elemental selenium and sodium selenite; Se 0 =Indicates the addition of elemental selenium, Se indicates the addition of sodium selenite; the amounts of selenium were 5, 10, 20, and 40 μM, respectively. “*” indicates p < 0.05; “**” indicates p < 0.01; and “***” indicates p < 0.001.

[0074] Figure 5c is the OD value of rhizobium CCBAU 33470 after 96 h of culture in TY liquid culture medium. 600nm Results. CK is the control, indicating no addition of elemental selenium and sodium selenite; Se 0 =Indicates the addition of elemental selenium, Se indicates the addition of sodium selenite; the amounts of selenium were 5, 10, 20, and 40 μM, respectively. “*” indicates p < 0.05; “**” indicates p < 0.01; and “***” indicates p < 0.001. DETAILED DESCRIPTION

[0075] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0076] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0077] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0078] The data in the following examples were processed using GraphPad Prism 9.5 statistical software. The experimental results were expressed as mean ± standard deviation, and the significance of the differences was tested using One-way ANOVA.

[0079] Example 1: Isolation, molecular identification and preservation of strain CGMCC No. 33109

[0080] 1. Isolation of organophosphate-solubilizing strain CGMCC No.33109

[0081] Soybean rhizosphere soil was collected from the field in Sunlaoying, Woyang County, Anhui Province (116°5'40.344" E, 33°32'32.212" N). The specific method was as follows: healthy soybean plants were selected and dug out with a shovel without damaging the soybean roots. The excess soil was shaken off and the rhizosphere soil was collected in a sterile sample bag. The bag was labeled, stored in an ice box, and brought back to the laboratory for processing on the same day.

[0082] Weigh 5 g of soybean rhizosphere soil sample and place it in a conical flask filled with 50 mL of sterile deionized water. Oscillate it in a shaker at 28°C and 180 rpm for 30 minutes to mix it thoroughly. Let it stand for 30 minutes, take the supernatant and perform gradient dilution. Take 10 -1 to 10 -5 100 μL of each gradient dilution was applied to Montana medium (to screen for bacteria that can solubilize organophosphate) and incubated upside down in a 28°C incubator for 3 days. Single colonies with phosphate-solubilizing properties were selected from the plates. This present invention only details the molecular identification and preservation of the strain originally designated GY-C-Y7 (with CGMCC accession number No. 33109), as well as the subsequent methods and applications for synthesizing elemental selenium using this strain.

[0083] 2. Molecular identification of strain CGMCC No.33109

[0084] 1. Amplification and sequencing of the 16S rRNA gene

[0085] Forward primer 27F sequence: 5′-AGGAGGTGATCCAGCCGCA-3′ (SEQ ID NO: 3);

[0086] Reverse primer 1492R sequence: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 4).

[0087] Colony PCR reaction system: 2×Taq PCR StarMix (Dye): 15 μL, forward primer 27F (10 μM): 1 μL, reverse primer 1492R (10 μM): 1 μL, ddH2O: 13 μL, template DNA: a small amount of the aforementioned single colony cells.

[0088] PCR amplification program: initial denaturation at 94°C for 2 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, and extension at 72°C for 1.5 min; and final extension at 72°C for 5 min.

[0089] After the PCR products were qualified by agarose gel electrophoresis, they were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The obtained 16S rDNA sequence (SEQ ID NO: 1) was first aligned on NCBI, and closely related sequences were found and downloaded. Then, a phylogenetic tree was constructed using MEGA software based on the Neighbor-Joining method.

[0090] The results are as follows Figure 1 The 16S rDNA sequence of CGMCC No.33109 strain is similar to that of Citrobacter arsenatis LY-1 T The 16S rDNA sequence (MK262983) of CGMCC No. 33109 was the highest, reaching 99.9%, which exceeded the threshold of 95% for bacterial attribution. Therefore, the strain CGMCC No. 33109 belonged to the genus Citrobacter.

[0091] 2. Amplification and Sequencing of the Housekeeping Gene recN

[0092] Forward primer recN-F sequence: 5′-ATGTTGGCACAACTGACCAT-3′ (SEQ ID NO: 5);

[0093] Reverse primer recN-R sequence: 5'-TTATGCCGCCAGCAGTTCT-3' (SEQ ID NO: 6).

[0094] Colony PCR reaction system: 2×Taq PCR StarMix (Dye): 15 μL, forward primer recN-F (10 μM): 1 μL, reverse primer recN-R (10 μM): 1 μL, ddH2O: 13 μL, template DNA: a small amount of the aforementioned single colony cells.

[0095] PCR amplification program: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 1.6 min, 30 cycles; final extension at 72°C for 5 min to terminate the PCR reaction.

[0096] After the PCR products were qualified by agarose gel electrophoresis, they were sent to Beijing Qingke Biotechnology Co., Ltd. for sequence determination.

[0097] The obtained recN sequence (SEQ ID NO: 2) was first aligned on NCBI, and the recN sequence of the representative species belonging to the genus Citrobacter was found and downloaded. Then, a phylogenetic tree was constructed using MEGA software based on the Neighbor-Joining method.

[0098] The results are as follows Figure 2 The recN sequence of strain CGMCC No.33109 is similar to that of Citrobacter arsenatis LY-1. T The recN sequence of CP037864 was the highest, reaching 99.6%, which exceeded the threshold of 97% for bacterial species identification. Therefore, strains CGMCC No.33109 and LY-1 T The two strains are of the same species, Citrobacter arsenatis.

[0099] 3. Deposit of strain CGMCC No.33109

[0100] Based on the homology analysis results of the above-mentioned 16S rDNA sequence and the housekeeping gene recN sequence, the strain isolated and purified in step 1 was identified as Citrobacter arsenatis, and the original strain number was GY-C-Y7. The strain GY-C-Y7 was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 18, 2024, with a deposit number of CGMCC No. 33109.

[0101] Example 2: Growth of CGMCC No. 33109 strain on Montana organophosphate medium and determination of its ability to dissolve lecithin

[0102] LB broth medium: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and distilled water to 1 L. For solid culture medium, 15 g / L of agar powder may be added. pH = 7.0. Autoclave at 121°C for 25 min.

[0103] Montgina organophosphate medium: glucose 10 g, ammonium sulfate 0.5 g, manganese sulfate 0.03 g, sodium chloride 0.3 g, potassium chloride 0.3 g, soybean lecithin 0.2 g, yeast extract 0.4 g, calcium carbonate 5 g, ferrous sulfate heptahydrate 0.03 g, agar powder 15 g, distilled water to 1 L; pH = 7.0-7.5; autoclave at 115°C for 20 min.

[0104] A single colony of CGMCC No.33109 strain grown on LB solid medium was inoculated into 5 ml LB liquid test tube medium and cultured at 200 rpm and 37°C for 12 h. The absorbance value of the bacterial solution was adjusted using sterile LB liquid medium. 600nm = 1.0. 2 μl of bacterial solution was spotted onto a solid plate of Montana organophosphate medium and incubated upside down at 37°C. The colony size and the size of the transparent phosphate-soluble zone around the colony were observed on the 3rd and 8th days, respectively.

[0105] The results are as follows Figure 3 As shown, the CGMCC No. 33109 strain can grow on the Montkina organophosphate medium and dissolve the lecithin in the Montkina organophosphate medium to produce a transparent phosphate-dissolving circle. The left picture shows the result after 3 days of culture, and the right picture shows the result after 8 days of culture.

[0106] Example 3: Extraction and purification of red elemental selenium synthesized by strain CGMCC No. 33109

[0107] Preparation of sodium selenite stock solution: Take 3.46g of sodium selenite (Na2SeO3, produced by Sinopharm Chemical Reagent Co., Ltd., chemically pure, batch number: 20240927) and dissolve it in 10mL of ultrapure water. After thorough mixing, dilute the volume to 20mL with ultrapure water, and then filter and sterilize with a 0.22μM sterile filter. Dispense into sterile 1.5mL sterile microcentrifuge tubes and store in a -20℃ refrigerator until used.

[0108] 1) Seed liquid culture: A single colony of strain CGMCC No. 33109 was inoculated into 5 mL of LB liquid medium and cultured at 37° C. and 200 rpm for 12 h.

[0109] 2) Expansion of bacterial culture and synthesis of red elemental selenium: The seed solution was adjusted using a spectrophotometer to obtain the OD 600nm = 1.0, add the seed solution to 300 mL of LB liquid medium at a 2% inoculum volume ratio (e.g., 2 mL of seed solution: 100 mL of LB liquid medium), and add sodium selenite solution to the medium to obtain a culture solution with a final sodium selenite concentration of 5 mM. Incubate at 37°C, 200 rpm for 18 h.

[0110] 3) Extraction and purification of red elemental selenium: Collect 300 mL of bacterial culture medium in the above step 2), collect the precipitate (bacteria) by centrifugation at 25°C and a centrifugal force of 8000g for 5 min, and wash the collected precipitate three times with phosphate buffered saline (PBS); resuspend the washed precipitate in 20 mL of PBS buffer, weigh 0.1 g of lysozyme and mix it thoroughly, and then incubate the mixture at room temperature for 30 min; ultrasonically disrupt the sample on ice, and the treatment conditions are 3 seconds on and 5 seconds off, 250w for a total of 160 cycles; after treatment, the suspension is centrifuged at 4°C and a centrifugal force of 8000g for 5 min. The suspension was washed three times with ddH2O to remove the residual white precipitate and obtain the sample, which was then resuspended in 5 mL of sterile water. 10 mL of 80% sucrose solution was added to the suspension and centrifuged at 4°C and 12000 g for 30 min to collect the bottom precipitate. The suspension was then washed four times with sterile water (12000 g for 5 min each time). The sample obtained from the fourth centrifugation was resuspended in sterile water and dispensed into 1.5 mL microcentrifuge tubes. The sample was centrifuged at 4°C and 12000 g for 5 min to collect the bottom precipitate. The purified red elemental selenium was placed in a freeze-drying vacuum dryer for 12 h. The treated sample was as shown in FIG. Figure 4 As shown, red precipitate was visible at the bottom, and the sample was stored in a -80°C refrigerator.

[0111] Example 4: Determination of the concentration of red elemental selenium synthesized by strain CGMCC No. 33109

[0112] 1. Drawing of the standard curve of molar content of selenium

[0113] Preparation method of 0.1mM sodium selenite solution: Dissolve 1.7295mg of sodium selenite (Na2Se03) in 90mL of ultrapure water, mix thoroughly, and then dilute to 100mL to obtain 0.1mM sodium selenite solution. Then, prepare sodium selenite solutions of different micromolar concentrations (μmol) according to Table 1.

[0114] Table 1. Preparation methods of Na2SeO3 with different micromolar numbers (μmol)

[0115]

[0116] Take 100 μL of the above Na2SeO3 solution containing 0.5-10 μmol of Na2SeO3 and add it to the empty centrifuge tubes prepared in advance, and then add 250 μL of HN2OH·HCl to each centrifuge tube to ensure that Na2SeO3 can be completely converted into elemental selenium (Se 0 After the reaction is complete, add 1 mL of 1 M Na2S solution to each centrifuge tube to make Se 0 After fully reacting with Na2S, the absorbance of the solution at 500nm (OD 500nm ), each sample was repeated three times. 0 The molar number (μmol) is on the X axis, and the absorbance value (OD 500nm ) is used as the Y-axis to draw the standard curve.

[0117] 2. Determination of elemental selenium concentration

[0118] Resuspension: The freeze-vacuum-dried red elemental selenium obtained in Example 3 was thoroughly resuspended in 1 mL of sterile water to obtain a resuspension. According to Table 2, the required volume of resuspension was taken and the required volume of sterile water was added to obtain four different 350 μL red elemental selenium suspensions with different red elemental selenium contents.

[0119] Table 2. Preparation of red elemental selenium solutions with different volumes

[0120] Amount of resuspension required (μL) Amount of sterile water required (μL) Red elemental selenium suspension (μL) 350 0 350 150 200 350 50 300 350 10 340 350

[0121] 1 mL of 1 M Na2S solution was added to each of the four 350 μL red elemental selenium solutions, and the mixture was gently shaken to fully dissolve and mix. The mixture was allowed to stand at room temperature for 1 h, and then the absorbance at 500 nm (OD) was measured by UV spectrophotometer. 500nm ), and finally, the red elemental selenium content of the red elemental selenium suspension corresponding to the absorbance value is calculated according to the standard curve formula.

[0122] Example 5: Red elemental selenium improves the drought stress tolerance of Rhizobium CCBAU 33470

[0123] Rhizobium CCBAU 33470 is derived from the following non-patent document "Zheng Wentao. Study on the genetic diversity of Chinese milk vetch and its relationship with environmental factors [D]. China Agricultural University", which is publicly available from China Agricultural University. This biological material is only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0124] Preparation method of TY liquid medium for culturing Rhizobium CCBAU 33470: 3 g yeast powder, 5 g tryptone, 0.6 g CaCl2, and deionized water to 1 L, pH 7.0. Sterilize at 121°C for 20 min.

[0125] Preparation method of TY solid medium for culturing Rhizobium CCBAU 33470: 3 g yeast powder, 5 g tryptone, 0.6 g CaCl2, 15 g agar powder, and deionized water to 1 L, pH 7.0. Sterilize at 121°C for 20 min.

[0126] Preparation method of TY liquid medium containing 15% PEG-6000 solution: According to the above TY liquid medium formula, add PEG-6000 with a final concentration of 15%, sterilize at 121°C for 20 minutes, and cool to below room temperature before use.

[0127] Different concentrations of PEG-6000 were added to the culture medium or water to simulate drought stress. The higher the concentration, the stronger the drought stress. PEG-6000 was purchased from Solarbio, with the product number: P8250.

[0128] The experiment was repeated three times, and each repetition was as follows:

[0129] 5μM elemental selenium group (i.e. Figure 5a 、 Figure 5b or Figure 5c Medium 5Se 0 ) : Rhizobium CCBAU 33470 was inoculated into TY liquid medium and cultured at 28°C in a shaker at 180 rpm for 72 h. The culture was centrifuged at 6000 rpm for 3 min to collect the cells. The pellet was then washed twice with sterile saline (0.85% NaCl) and resuspended in saline. The OD was then adjusted using a spectrophotometer. 600nm =1.0. With initial OD 600nm =0.02 The bacterial liquid was inoculated into TY liquid medium containing 15% PEG-6000 solution, and a selenium solution (obtained in Example 4) was added to the medium to make the final concentration of selenium in the medium 5 μM. The culture medium was placed in a shaker at 28°C and cultured at 180 rpm. 200 μL of the sample was taken at 48 h, 72 h, and 96 h, and the absorbance at 600 nm (OD) was measured using a microplate reader. 600nm ).

[0130] 10μM elemental selenium group (i.e. Figure 5a 、 Figure 5b or Figure 5c Medium 10Se 0): The difference between this group and the 5 μM elemental selenium group is that the final concentration of elemental selenium in the culture medium is 10 μM, and the rest of the operations are the same as those of the 5 μM elemental selenium group.

[0131] 20μM elemental selenium group (i.e. Figure 5a 、 Figure 5b or Figure 5c Medium 20Se 0 ): The difference between this group and the 5 μM elemental selenium group is that the final concentration of elemental selenium in the culture medium is 20 μM, and the rest of the operations are the same as those of the 5 μM elemental selenium group.

[0132] 40μM elemental selenium group (i.e. Figure 5a 、 Figure 5b or Figure 5c Medium 40Se 0 ): The difference between this group and the 5 μM elemental selenium group is that the final concentration of elemental selenium in the culture medium is 40 μM, and the rest of the operations are the same as those of the 5 μM elemental selenium group.

[0133] 5 μM sodium selenite group (i.e. Figure 5a 、 Figure 5b or Figure 5c 5Se): This group differs from the 5μM elemental selenium group in that sodium selenite solution is used instead of elemental selenium solution and the final concentration of sodium selenite in the culture medium is maintained at 5μM. Other operations are the same as those of the 5μM elemental selenium group.

[0134] 10 μM sodium selenite group (i.e. Figure 5a 、 Figure 5b or Figure 5c 10 μM sodium selenite): The difference between this group and 5 μM sodium selenite is that the final concentration of sodium selenite in the culture medium is 10 μM, and the rest of the operations are the same as those of the 5 μM sodium selenite group.

[0135] 20 μM sodium selenite group (i.e. Figure 5a 、 Figure 5b or Figure 5c 20 μM sodium selenite): The difference between this group and 5 μM sodium selenite is that the final concentration of sodium selenite in the culture medium is 20 μM, and the rest of the operations are the same as those of the 5 μM sodium selenite group.

[0136] 40 μM sodium selenite group (i.e. Figure 5a 、 Figure 5b or Figure 5c 40 μM sodium selenite): The difference between this group and 5 μM sodium selenite is that the final concentration of sodium selenite in the culture medium is 40 μM, and the rest of the operations are the same as those of the 5 μM sodium selenite group.

[0137] CK group: The difference between this group and 5 μM sodium selenite is that the final concentration of sodium selenite in the culture medium is 0 μM and the final concentration of elemental selenium is 0 μM. Other operations are the same as those of the 5 μM sodium selenite group.

[0138] The results are as follows Figure 5a、 Figure 5b and Figure 5c Under the stress of 15% PEG-6000, in the control treatment (CK) without selenium, rhizobia were cultured for 48-96 hours, and the absorbance of the culture solution (OD 600nm ) is the lowest; after adding elemental selenium, the absorbance of the rhizobium culture solution can be significantly increased after culturing for more than 72 hours (OD 600nm ), among which the effect of adding 40μM elemental selenium was the most significant. The addition of different concentrations of sodium selenite had little effect on rhizobia under drought stress conditions, with no significant difference. These results show that the addition of elemental selenium to rhizobia culture solution containing 15% PEG-6000 and simulating drought conditions can help rhizobia to tolerate drought. Under these conditions, the growth of rhizobia was promoted, the density of rhizobia increased, and the absorbance value of the culture solution (OD 600nm ) is therefore increased.

[0139] SEQ ID NO: 1

[0140]

[0141] SEQ ID NO:2

[0142]

[0143] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A strain of Citrobacter arsenatis, strain numbered GY-C-Y7, which has a registration number of CGMCC No. 33109 at the General Microbiology Center of the China Culture Collection Administration.

2. A composition characterized in that The composition contains the Citrobacter arsenate described in claim 1.

3. The composition according to claim 2, characterized in that The composition is a culture, and the culture is a substance obtained by culturing the Citrobacter arsenate in a microbial culture medium.

4. The composition according to claim 2, characterized in that The composition is a bacterial agent.

5. The composition according to claim 2, characterized in that The composition is a preparation for dissolving organic phosphorus or a preparation for preparing red elemental selenium.

6. Application, characterized in that, Use of the Citrobacter arsenate described in claim 1 in any of the following: A1) Dissolved organic phosphorus; A2) Preparation of products for dissolving organic phosphorus; A3) Reduce sodium selenite to red elemental selenium; A4) Prepare the product of reducing selenite to red elemental selenium.

7. Application, characterized in that, Use of the composition according to any one of claims 2 to 5 in any of the following: B1) dissolved organic phosphorus; B2) Preparation of products for dissolving organic phosphorus; B3) Reduce sodium selenite to red elemental selenium; B4) Prepare the product of reducing sodium selenite to red elemental selenium.

8. A method for preparing red elemental selenium, characterized in that: The method comprises culturing the Citrobacter arsenate described in claim 1 in a culture medium containing sodium selenite, collecting the bacterial cells, and extracting and purifying red elemental selenium from the bacterial cells.

9. A method for culturing Citrobacter arsenate, characterized in that: The Citrobacter arsenate is the Citrobacter arsenate according to claim 1, and the method comprises the step of culturing the Citrobacter arsenate in a culture medium for culturing microorganisms.

10. A method for preparing a composition, characterized in that The composition is the composition according to any one of claims 2 to 4, and the method comprises the step of using the Citrobacter arsenate described in claim 1 as a component of the composition.

11. A method for preparing a fermentation product, comprising culturing the Citrobacter arsenate described in claim 1 to obtain a fermentation product, wherein the fermentation product has the function of dissolving organic phosphorus.

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