Ensifer morelensis R2-120 and application thereof

Abstract

The invention relates to the technical field of microbiology and selenium polluted environment restoration, and provides an ensifer morelensis R2-120 strain and an application thereof. The bacterial strain R2-120 is ensifer morelensis capable of efficiently synthesizing volatile selenium, and the preservation number of the bacterial strain R2-120 is GDMCC No: 63730. The strain R2-120 has relatively strong tolerance to selenate and selenite, and a plate test shows that the R2-120 can tolerate at least 200 mM of selenate and 60 mM of selenite. The strain R2-120 can metabolize selenite and convert the selenite into nano-selenium, and can convert the selenite and the nano-selenium into volatile selenium. The invention also establishes a method for removing selenium in the water body by using R2-120 in the bioreactor, and the method can be used for bioremediation of selenium-polluted water body and soil, treatment of selenium-containing wastewater and recycling of selenium, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and selenium-contaminated environmental remediation technology, specifically to a strain of Ensifer morelensis R2-120 and its applications. Background Technology

[0002] Selenium is an essential trace element for the normal life activities of humans and animals, and it has important applications in industries such as metallurgy and ceramics. These selenium-related industrial processes discharge high-concentration selenium-containing wastewater. Direct discharge of untreated wastewater into the environment causes serious selenium pollution and wastes selenium resources. Compared with physicochemical remediation methods, microbial treatment of selenium-containing wastewater is a safe, efficient, and environmentally friendly remediation approach. Studies have shown that various microorganisms have the ability to reduce selenate or selenite to synthesize nano-selenium and volatile methylselenate. Nano-selenium and volatile selenium synthesized by microorganisms are easily separated and recovered from the aqueous phase. Furthermore, nano-selenium is a bacteriostatic agent with excellent biological activity in medicine and animal feed. Volatile methylselenate can be introduced into nitric acid through a tail gas capture device, and the resulting methylselenic acid is a novel antitumor drug.

[0003] Ensifer belongs to the family Rhizobiaceae and is a Gram-negative bacterium. This genus currently has 24 validly published species and possesses a strong ability to fix nitrogen. [1] Currently, there are no reports on *Strombus haematocephala* utilizing selenate or selenite to generate nano-selenium or methylselenium. Research on the biotransformation of selenium by *Strombus haematocephala* is beneficial for further developing and utilizing new selenium-repairing strains, and for the large-scale production of safe and efficient nano-selenium additives, as well as methylselenic acid drugs with excellent anti-cancer properties. Summary of the Invention

[0004] The purpose of this invention is to provide a strain of *Ensifer morelensis* R2-120 that can metabolize selenite into nano-selenium and convert selenite and nano-selenium into volatile selenium, and its applications, particularly in the remediation of selenium-contaminated environments and the preparation of methylselenic acid.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides a strain R2-120, isolated and purified from selenium-rich soil in Enshi, Hubei Province, capable of efficiently synthesizing volatile selenium. It is classified and named *Ensifer morelensis* and is currently deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China (Institute of Microbiology, Guangdong Academy of Sciences), accession number GDMCC No: 63730, deposited on August 11, 2023.

[0006] Secondly, the present invention provides a microbial preparation containing *Streptococcus faecium* R2-120.

[0007] Thirdly, this invention provides the application of *Streptococcus faecium* R2-120 in the remediation of selenium-polluted environments and the preparation of nano-selenium.

[0008] Fourthly, the present invention provides a method for the biosynthesis of nano-selenium, wherein *Streptococcus faecium* R2-120 is inoculated into a culture medium containing selenite and / or selenate, and after being cultured for a period of time, nano-selenium is isolated and purified from the culture medium.

[0009] In the culture medium containing selenite and / or selenate, the initial selenium concentration is 2-60 mM, preferably 2-20 mM, more preferably 2 mM, 5 mM, 10 mM, 20 mM, and most preferably 5 mM.

[0010] Fifthly, the present invention provides a method for synthesizing volatile selenium, wherein *Strombus fasciatus* R2-120 is inoculated into a culture medium containing selenite and / or nano-selenium, and after being cultured for a period of time, the generated volatile selenium is collected;

[0011] The volatile selenium includes dimethylselenoketone (DMSeO2) and dimethyldiselenoether (DMDSe).

[0012] Furthermore, in the culture medium containing selenite and / or nano-selenium, the initial selenium concentration is 0.125-0.5 mM, preferably 0.125 mM, 0.25 mM, or 0.5 mM, and more preferably 0.5 mM.

[0013] Sixthly, the present invention provides the application of the above method in the preparation of methylselenic acid.

[0014] The volatile selenium produced by the above method is passed into a capture solution containing an oxidant, so that the volatile selenium is oxidized into water-soluble methylselenic acid.

[0015] The oxidizing agent includes, but is not limited to, nitric acid or hydrogen peroxide.

[0016] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0017] The *Sclerotium spp.* R2-120 strain of this invention exhibits strong tolerance to selenate and selenite. Plate tests show that R2-120 can tolerate at least 200 mM selenate and 60 mM selenite. Strain R2-120 can metabolize selenite into nano-selenium and convert both selenite and nano-selenium into volatile selenium. This invention also establishes a method for removing selenium from water using R2-120 in a bioreactor. This method can be used for the bioremediation of selenium-contaminated water and soil, as well as for the treatment and recovery of selenium-containing wastewater, showing broad application prospects. Attached Figure Description

[0018] Figure 1 The colony and cell morphology characteristics of strain R2-120 of this invention are shown.

[0019] Figure 2 This is a phylogenetic tree of the 16S rRNA gene of strain R2-120 of this invention. Note: Bootstrap values ​​greater than 50% are shown in the figure. The numbers in parentheses are the GenBank accession numbers for the strain's 16S rRNA gene sequence, and the scale bar represents a 1% nucleotide substitution rate.

[0020] Figure 3 This is a phylogenetic tree comparing the genomes of strain R2-120 of this invention with other model species of the genus *Cymbidium*. Note: The phylogenetic tree was constructed using FastME 2.1.6.1. [2] The branch length scale was calculated using the GBDP evolutionary distance formula d5, and the displayed branch values ​​are from 100 GBDP pseudo-bootstrap values. The average branch support is 93.4%. The scale represents a 2% nucleotide substitution rate.

[0021] Figure 4 The tolerance of strain R2-120 to selenate and selenite in a preferred embodiment of the present invention.

[0022] Figure 5 The yield of nano-selenium synthesized by strain R2-120 reducing Se(IV) in a preferred embodiment of the present invention.

[0023] Figure 6 The physicochemical characteristics of the synthesis of nano-selenium by reducing Se(IV) with strain R2-120 in a preferred embodiment of the present invention are described.

[0024] Figure 7 The volatilization efficiency of strain R2-120 for selenite and nano-selenium in a preferred embodiment of the present invention.

[0025] Figure 8 This invention relates to the identification of volatile selenium synthesized by strain R2-120 in a preferred embodiment of the present invention.

[0026] Figure 9In a preferred embodiment of the present invention, methylselenic acid is synthesized using strain R2-120 in a fermenter. Detailed Implementation

[0027] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0028] Example 1: Isolation and Identification of Cyclosporium R2-120 strain

[0029] 1. Strains Isolation

[0030] Rhizosphere soil of *Capsella bursa-pastoris*, a selenium-accumulating plant, was collected from selenium-rich soil in Enshi, Hubei Province. 5 g of the soil was added to 95 mL of sterile physiological saline and extracted by shaking at 25℃ and 150 rpm for 30 min. The soil suspension was then diluted 10 times using a 10-fold serial dilution method. 1 -10 6 100 μL of each gradient dilution was evenly spread onto NA plates containing 50 mM selenite (NA medium: peptone 10.0 g / L, beef extract 3.0 g / L, sodium chloride 5.0 g / L, agar 15.0 g / L, pH 7.3±0.1, sterilized at 121℃ for 15 min), and incubated at 28℃ for 2 days to obtain selenite-tolerant strains. From these strains, a strain R2-120 capable of reducing selenite was isolated and purified.

[0031] 2. Analysis of colony and cell morphology characteristics

[0032] R2-120 was cultured on TSA plates (TSA medium: tryptone 15.0 g / L, soybean peptone 5.0 g / L, sodium chloride 5.0 g / L, agar 18.0 g / L, pH 7.3±0.1, sterilized at 121℃ for 15 min) for 2 days. Colonies were observed to be round, white, with a diameter ranging from 1.0 to 2.0 mm, with neat and glossy edges. Transmission electron microscopy revealed rod-shaped cells with flagella, 0.8–2.0 μm long and 0.5–1.2 μm wide. Figure 1 ).

[0033] 3. Phylogenetic analysis of 16S rRNA genes

[0034] Genomic DNA was extracted from strain R2-120 using a bacterial DNA extraction kit (DL111-01, BMamp). The 16S rRNA gene was amplified using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The PCR system (25 μL) consisted of: 12.5 μL of mixed enzyme; 1.0 μL each of primers (10 μM); 1.0 μL of DNA template (100 ng / μL); and 9.5 μL of ddH2O. The PCR reaction program was as follows: 94℃ for 10 min; 94℃ for 40 s, 56℃ for 40 s, 72℃ for 40 s, 30 cycles; 72℃ for 10 min. The PCR product was purified and sequenced. The obtained sequence was compared with the 16S rRNA gene sequence in the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) using the BLAST program. The results showed that the 16S rRNA gene sequence of the strain (SEQ ID NO:1) was similar to that of Ensifer morelensis Lc04. T The similarity was the highest, reaching 99.86%. The phylogenetic tree of the 16S rRNA gene of strain R2-120, constructed using the Mega software with the neighbor-joining method and the Kimura2-parameter model, is shown below. Figure 2 strain R2-120 is related to Ensifermorelensis LMG 21331 T They clustered on one branch.

[0035] 4. Genome sequencing and phylogenetic analysis

[0036] Whole-genome sequencing of strain R2-120 was performed using the Illumina NovaSeq PE150 platform, yielding a genome size of 6.6 Mb and a GC content of 61.9 mol%. Species identification and phylogenetic tree construction were performed using the TYGS platform (https: / / tygs.dsmz.de). Figure 3 The results showed that R2-120 belongs to the genus *Ensifer*, and is related to *Ensifer morelensis* Lc04. T Those that cluster on one branch are the closest in kinship.

[0037] 5. Genome similarity analysis

[0038] Genome similarity analysis was performed on R2-120 and other type strains of the genus *Ensifer adhaerens*. Average Nucleotide Identity (ANI) was calculated using the JSpeciesWS platform (https: / / jspecies.ribohost.com / jspeciesws / #analyse), and digital DNA-DNA hybridization (dDDH) was calculated using GGDC Calculator 3.0 (http: / / ggdc.dsmz.de) (Table 1). After comparison, strain R2-120 was found to be similar to two strains in the NCBI database, *Ensifer adhaerens* Lc04. T And Ensifer adhaerens DSM 18131 T The ANI values ​​of (GCA_013283195.1 and GCA_017873015.1) are 97.6% and 97.7%, respectively, and the dDDH values ​​are 83.3% and 83.8%, respectively. The genomic similarity exceeds the species threshold, indicating that they are the same species.

[0039] Table 1. ANI and dDDH values ​​of strain R2-120 and other type strains of the genus *Cymbidium*.

[0040] strain Size (Mb) GC content (mol%) <![CDATA[dDDH, d4]]> ANIb Ensifer sp. R2-120 6.7 61.9 * * <![CDATA[Ensifer adhaerens Casida A T ]]> 7.3 62.3 25.5 81.0 <![CDATA[Ensifer alkalisoli YIC4027 T ]]> 6.0 62.2 22.8 77.5 <![CDATA[Ensifer americanum CFNEI 156 T ]]> 6.7 62.3 23.0 77.8 <![CDATA[Ensifer arboris LMG 14919 T ]]> 6.8 62.0 22.8 77.4 <![CDATA[Ensifer aridi LMR001 T ]]> 6.6 61.7 22.9 77.9 <![CDATA[Ensifer canadensis T173 T ]]> 8.0 61.0 35.5 86.7 <![CDATA[Ensifer fredii NBRC 14780 T ]]> 6.6 62.3 23.2 78.1 <![CDATA[Ensifer garamanticum LMG 24692 T ]]> 6.8 61.3 23.1 78.0 <![CDATA[Ensifer glycinis CCBAU 23380 T ]]> 6.0 62.4 23.6 78.4 <![CDATA[Ensifer kostiense DSM 13372 T ]]> 6.3 61.7 22.8 77.3 <![CDATA[Ensifer kummerowiae CCBAU 71714 T ]]> 6.6 62.1 23.1 77.7 <![CDATA[Ensifer medicae USDA1037 T ]]> 6.5 61.2 22.1 76.7 <![CDATA[Ensifer meliloti NBRC 14782 T ]]> 6.7 62.1 23.1 77.8 <![CDATA[Ensifer mexicanum ITTG R7 T ]]> 7.1 61.5 23.7 77.9 <![CDATA[Ensifer morelensis DSM 18131 T ]]> 6.8 62.0 83.3 97.7 <![CDATA[Ensifer morelensis Lc04 T ]]> 7.1 61.7 83.8 97.6 <![CDATA[Ensifer numidicum CIP 109850 T ]]> 6.7 60.7 23.0 78.0 <![CDATA[Ensifer oleiphilus HO-A22 T ]]> 6.8 61.7 45.9 91.0 <![CDATA[Ensifer psoraleae CCBAU 65732 T ]]> 7.4 61.2 23.2 78.0 <![CDATA[Ensifer saheli LMG 7837 T ]]> 6.0 63.6 23.7 78.5 <![CDATA[Ensifer sesbaniae CCBAU 65729 T ]]> 6.9 62.1 25.6 80.9 <![CDATA[Ensifer sojae CCBAU 05684 T ]]> 6.1 62.0 23.0 77.8 <![CDATA[Rhizobium leguminosarum USDA 2370 T ]]> 7.8 60.6 20.8 73.1

[0041] There are inconsistencies in the species names annotated in the NCBI and ENA databases for Ensifer sp. Lc04 and Ensifer sp. DSM 18131. The NCBI database annotates it as Ensifer adhaerens, but both are different from the type strain of Ensifer adhaerens, Ensifer adhaerens Casida A. T The ANI and dDDH of (GCA_000697965.2) are both below the species threshold, indicating an error in the species name annotation. However, in the ENA database, both are annotated as *Ensifer morelensis*. Comparison with the pattern strains in the LPSN database shows that the correct classification for strains Lc04 and DSM 18131 should be *Ensifer morelensis* Lc04. T and Ensifer morelensis DSM 18131 T .

[0042] 6. Stress Resistance Experiment: Fresh colonies of strain R2-120 were inoculated onto TSA plates and cultured at 4, 10, 20, 28, 30, 32, 34, 37, 40, and 42℃ for 4 days, respectively, and the growth of the strain was observed. Single colonies of R2-120 were picked and inoculated into TSB test tubes (TSB medium: 15.0 g / L tryptone, 5.0 g / L soybean peptone, 5.0 g / L sodium chloride, pH 7.3±0.1, sterilized at 121℃ for 15 min), and activated by shaking at 28℃ and 150 rpm for 12 h, adjusting OD... 600 The pH was set at 0.8 as the seed culture. NaCl was added to TSB medium to prepare saline media containing 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% (w / v). The pH of the TSB medium was adjusted to 4, 5, 6, 7, 8, 9, 10, and 11 using citric acid (0.1 M) and NaOH (0.1 M), respectively. Seed cultures of strain R2-120 were inoculated into saline TSB medium and TSB media at different pH values, and cultured at 28℃ and 150 rpm for 4 days. Growth was observed. The results showed that strain R2-120 had higher tolerance to NaCl than closely related bacteria, tolerating 4% NaCl. The growth temperature range of R2-120 was 4-37℃, and the growth pH range was 6-11 (Table 2).

[0043] Table 2 Growth and stress resistance indicators of R2-120 and reference strains

[0044] index R2-120 <![CDATA[Ensifer morelensis Lc04 T ]]> Temperature tolerance (°C) 4-37 28 NaCl tolerance 1-4% 0-2% pH tolerance 6-11 5-10

[0045] According to the "Handbook for Identifying Common Bacteria" [3] The routine microbial identification methods were used to perform physiological and biochemical identification on strain R2-120. The results showed that strain R2-120 is a facultative anaerobe and lacks motility. It is oxidase-positive, catalase-negative, does not produce DNase, and is not hemolytic.

[0046] Based on the morphological characteristics, phylogenetic analysis, ANI and dDDH calculations, and physiological and biochemical characteristics of R2-120, strain R2-120 was identified as *Ensifer morelensis*. This strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center on August 11, 2023, with accession number GDMCC No. 63730.

[0047] Example 2: Tolerance of strain R2-120 to selenate and selenite

[0048] Melt and cool LB solid medium (LB medium: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, solid medium plus agar 18 g / L, pH 7.0-7.2, sterilized at 121℃ for 20 min) to approximately 60℃. Add sterile sodium selenite solution (filtered and sterilized), gently shake to mix, and pour into plates to prepare plates containing 0-200 mM (as Se) sodium selenite or 0-100 mM sodium selenate. Serially dilute R2-120 seed culture with sterile physiological saline in 10-fold increments, and add 2.5 μL of each dilution to a selenium-containing plate. After air-drying under sterile conditions, incubate at 28℃ for 48 h.

[0049] The growth of strain R2-120 on selenate and sodium selenite solid media is shown in the figure. Figure 4 The results showed that R2-120 could tolerate at least 200 mM selenate and 60 mM selenite. The cells in the high-concentration selenate treatment (≥100 mM) and selenite treatment (≥20 mM) groups turned red, indicating that R2-120 could reduce selenate and selenite to red nano-selenium.

[0050] Example 3: The reducing ability of strain R2-120 to selenite and the biosynthesis of nano-selenium.

[0051] Add filtered and sterilized sodium selenite stock solution to 50 mL LB to achieve selenium concentrations of 2 mM, 5 mM, 10 mM, and 20 mM (as Se), and inoculate with 1% v / v R2-120 seed culture (OD). 600 After adding 0.8 g of the sample, it was placed at 28°C and 150 rpm for shaking incubation for 48 h.

[0052] Using sodium sulfide spectrophotometry [4] Determination of nano-selenium yield: Centrifuge 500 μL of the test bacterial solution at 12000 rpm for 5 min, resuspend in sterile physiological saline, centrifuge again and discard the supernatant. After washing 3 times, add 1 mL of freshly prepared 1 M Na2S solution, mix thoroughly, react for 1 h, centrifuge at 12000 rpm for 5 min, and measure the absorbance of the supernatant at a wavelength of 500 nm. Calculate the nano-selenium yield in the bacterial solution based on the nano-selenium standard curve. The results are shown in [Figure number missing]. Figure 5 Under treatment with 2-20 mM Se(IV), strain R2-120 was able to synthesize selenium nanoparticles, and the yield of selenium nanoparticles gradually increased. At a Se(IV) concentration of 5 mM, the yield and conversion rate of selenium nanoparticles reached the highest levels, reaching 2.89 mM and 57.7%, respectively.

[0053] Selenium nanoparticles synthesized by R2-120 were separated and purified as follows: The bacterial culture was centrifuged at 8000 rpm for 10 min. The collected red precipitate was washed three times with sterile physiological saline and resuspended in purified water to obtain a selenium nanoparticle suspension. The selenium nanoparticle suspension was transferred to an extraction column, and 0.8 times the volume of n-hexane was added. Extraction was performed four times, and the lower aqueous phase was collected. The mixture was centrifuged at 4000 rpm for 40 min, and the resulting precipitate was washed three times with sterile physiological saline. The precipitate was then freeze-dried to obtain pure selenium nanoparticle powder.

[0054] Example 4 Physicochemical characteristics of the synthesis of nano-selenium by reduction of Se(IV) by strain R2-120

[0055] Seed culture of R2-120 was inoculated into LB medium containing 5 mM Se(IV) and cultured at 28℃ and 150 rpm for 2 days, during which red selenium nanoparticles were observed to form. The physicochemical characteristics of the selenium nanoparticles synthesized by strain R2-120 were analyzed using transmission electron microscopy and X-ray energy dispersive spectroscopy.

[0056] Figure 6 The results showed that the selenium nanoparticles synthesized by R2-120 were nearly spherical with a diameter ranging from 150 to 300 nm, mainly distributed extracellularly, although intracellular selenium nanoparticles were also observed. EDS analysis of the R2-120 nanoparticles revealed characteristic peaks of selenium at 1.37 keV, 11.22 keV, and 12.50 keV, with a selenium mass ratio of 99.8%, indicating that the nanoparticles synthesized by the strain were indeed selenium nanoparticles.

[0057] Example 5: Volatilization efficiency of strain R2-120 for selenite and nano-selenium

[0058] Preparation of nano-selenium mother liquor: Using 1000 mL of 0.1 M ascorbic acid as a reducing agent and 100 mL of 1 wt% PVPK30 as a dispersant, 500 mL of 0.1 M sodium selenite was magnetically stirred at room temperature for approximately 20 min until the color of the reactants changed from colorless to red. The obtained SeNPs were centrifuged at 10000 g for 30 min and washed three times with deionized water. The purified SeNPs were resuspended in deionized water and filtered through a 0.22 μm filter membrane for sterilization to obtain the nano-selenium mother liquor, which was stored at 4℃. [5] The concentration of nano-selenium mother liquor was determined using the sodium sulfide method.

[0059] Inoculate 0.5 mL of R2-120 seed culture (OD) into a 150 mL shake flask containing 50 mL of LB medium. 600=0.8) and the corresponding volume of sodium selenite or nano-selenium stock solution (filtered and sterilized) were used to make initial selenium concentrations of 0.125, 0.25, and 0.5 mM (as Se), respectively. The mixture was then incubated at 28℃ and 150 rpm for 4 days with shaking. The decrease in total selenium in the bacterial culture after 4 days of incubation was measured using atomic fluorescence spectrometry (HG-AFS), and the selenium volatilization rate was calculated accordingly.

[0060] Selenium volatilization rate (%) = (Selenium added to bacterial solution - Remaining selenium in bacterial solution) / Selenium added to bacterial solution × 100%

[0061] Selenium content detection results showed that after 4 days of cultivation, the selenium content in the R2-120 culture medium decreased significantly, and a strong garlic odor was produced during the cultivation process. Within the selenium addition range of 0.125-0.5 mM, selenium volatilization increased with increasing initial selenium addition, reaching its highest level at 0.5 mM. Selenium volatilization for Se(IV) and SeNPs reached 0.35 mM and 0.30 mM, respectively, with volatilization rates of 68.8% and 58.5%. At a selenium addition level of 0.25 mg / L, the volatilization rates for Se(IV) and SeNPs were the highest, at 73.9% and 86.1%, respectively. Figure 7 ).

[0062] Example 6 Identification of volatile selenium synthesized by strain R2-120

[0063] Inoculate R2-120 seed culture (OD) at a 1% v / v inoculum. 600 =0.8) was inoculated into 3 mL LB headspace vials containing 0.5 mM sodium selenite or nano-selenium (calculated as selenium) and incubated at 28℃ with shaking at 150 rpm. After 96 h of incubation, the culture was analyzed by gas chromatography-mass spectrometry (GC-MS). [6] The selenium speciation of headspace gases was identified, and the results showed that ( Figure 8 R2-120 converts Se(IV) and SeNPs into DMSeO2 (dimethylselenoketone) and DMDSe (dimethyldiselenoether), without the formation of DMSe (dimethylselenoether). DMDSe has the highest abundance.

[0064] Example 7 Synthesis of methylselenic acid using strain R2-120 in a fermenter

[0065] Inoculate R2-120 seed culture (OD) at a 1% v / v inoculum. 600 =0.8) was inoculated into the fermenter. The culture medium in the fermenter was 6 L of sterile LB medium containing 5 mM Se(IV) (sodium selenite). The aeration rate of the experimental setup was 0.18 m 3Fermentation was carried out at 28±0.5℃ for 48 h at a pressure of 0.05-0.06 MPa and a stirring speed of 150 rpm. The tail gas was then piped into the concentrated nitric acid absorption solution for absorption. The identification of selenium species in the concentrated nitric acid absorption solution using high-performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS) showed that the main selenium species in the nitric acid capture solution was methylseleninic acid (MSA). Figure 9 ), is an oxidation product of DMDSe (dimethyl diselenide). [7] After collection, it can be further processed to produce anti-tumor drugs. R2-120 does not generate DMSe (dimethyl selenide), and only methylselenic acid was detected as a selenium-related product in the nitric acid capture solution, without interference from other selenium compounds (dimethyl selenide oxide), which shows great potential in the preparation of anticancer drugs.

[0066] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0067] References:

[0068] [1]Verástegui-Valdés MM, Zhang YJ, Rivera-Orduña FN, Cheng HP,Sui XH, Wang E T. Microsymbionts of Phaseolus vulgaris in acid and alkalinesoils of Mexico [J]. Systematic and Applied Microbiology, 2014, 37(8): 605-12.

[0069] [2]Lefort V, Desper R, Gascuel O. FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program [J]. MolecularBiology and Evolution, 2015, 32(10): 2798-800.

[0070] [3] Dong Xiuzhu, Cai Miaoying. Handbook of Systematic Identification of Common Bacteria [M]. Handbook of Systematic Identification of Common Bacteria, 2001.

[0071] [4]Biswas K C, Barton L L, Tsui W L, Shuman K, Gillespie J, Eze C S.A novel method for the measurement of elemental selenium produced bybacterial reduction of selenite [J]. Journal of Microbiological Methods,2011, 86(2): 140-4.

[0072] [5]Selmani A, Ulm L, Kasemets K, Kurvet I, Erceg I, Barbir R, Pem B,Santini P, Marion I D, Vinkovic T, Krivohlavek A, Sikiric M D, Kahru A, VrcekI V. Stability and toxicity of differently coated selenium nanoparticlesunder model environmental exposure settings [J]. Chemosphere, 2020, 250.

[0073] [6]Kagami T, Narita T, Kuroda M, Notaguchi E, Yamashita M, Sei K,Soda S, Ike M. Effective selenium volatilization under aerobic conditions andrecovery from the aqueous phase by Pseudomonas stutzeri NT-I [J]. WaterResearch, 2013, 47(3): 1361-8.

[0074] [7]Burra R, Pradenas G A, Montes R A, Vasquez C C, Chasteen T G.Production of dimethyl triselenide and dimethyl diselenenyl sulfide in theheadspace of metalloid-resistant Bacillus species grown in the presence ofselenium oxyanions [J]. Analytical Biochemistry, 2010, 396(2): 217-22.

Claims

1. Ensifer morelensis R2-120, accession number GDMCC No: 63730.

2. A microbial preparation containing the *Strombus* strain of claim 1.

3. The application of the *Strombus haematocephala* as described in claim 1 in the remediation of selenium-polluted environments and the preparation of nano-selenium.

4. A method for the biosynthesis of nano-selenium, characterized in that, The *Brachys lanceolata* of claim 1 was inoculated into a culture medium containing selenite, and after being cultured for a period of time, nano-selenium was isolated and purified from the culture medium.

5. The method according to claim 4, characterized in that, In the culture medium containing selenite, the initial selenium concentration is 2-60 mM, preferably 2-20 mM, more preferably 2 mM, 5 mM, 10 mM, or 20 mM, and most preferably 5 mM.

6. A method for synthesizing volatile selenium, characterized in that, The *Strombus haemolyticus* of claim 1 is inoculated into a culture medium containing selenite and / or nano-selenium, and after being cultured for a period of time, the volatile selenium produced is collected. The volatile selenium includes dimethylselenophenone and dimethyldiselenoether.

7. The method according to claim 6, characterized in that, In the culture medium containing selenite and / or nano-selenium, the initial selenium concentration is 0.125-0.5 mM, preferably 0.125 mM, 0.25 mM, or 0.5 mM, and more preferably 0.5 mM.

8. The use of the method of claim 6 or 7 in the preparation of methylselenic acid.

9. The application according to claim 8, characterized in that, The volatile selenium produced according to the method of claim 6 or 7 is passed into a capture solution containing an oxidant, such that the volatile selenium is oxidized to water-soluble methylselenic acid.

10. The application according to claim 9, characterized in that, The oxidant includes nitric acid or hydrogen peroxide.

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