Sphingopyxis sp. R3-92 and application thereof

Abstract

The invention relates to the technical field of microbiology and biological nano-selenium preparation, and provides a sphingomyza sp. R3-92 strain and application of the sphingomyza sp. R3-92 strain. The strain R3-92 is a new strain of sphingomyza sp capable of efficiently converting selenite into nano-selenium, the new strain is planned to be named as selenium reduction sphingomyza sp, and the preservation number of the new strain is GDMCC No: 63729. The strain R3-92 is a new strain of sphingomyza sp, is planned to be named as selenium reduction sphingomyza sp, and is named as selenium reduction sphingomyza sp. The strain R3-92 has relatively strong tolerance to selenate and selenite, and a plate culture test shows that the R3-92 can tolerate 100 mM of selenate and 80 mM of selenite. The invention also establishes a method for preparing a large amount of nano-selenium by using R3-92 in a bioreactor, and the method has obvious application effects in fertilizer, feed, selenium-rich functional food processing, health care products and medical products.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and bio-nano selenium preparation technology, specifically to a strain of Sphingopyxis sp. R3-92 and its applications. Background Technology

[0002] Selenium is a naturally occurring element with primary valence states of -2, 0, tetra, and hexavalent. Since the advent of the industrial age, the glass manufacturing industry has utilized selenium for coloring and decolorization, the metallurgical industry has used selenium dioxide to electrolyze manganese, and mining processes generate selenium dust and selenium-containing waste. Furthermore, selenium, as an essential trace element for humans and animals, has important applications in health products, feed, and fertilizers. The high-selenium wastewater generated by these industrial activities mainly contains selenate [Se(VI)] and selenite [Se(IV)], two highly toxic inorganic selenium compounds. Ingestion of high concentrations of selenium can cause selenium poisoning, leading to nail and hair loss, loss of appetite, reduced reproductive function, and weakened immunity. In contrast, nano-selenium is a low-toxicity, highly bioactive antibacterial agent with enormous application potential in the biomedical field and in food and feed additives. Therefore, using microorganisms to reduce selenate or selenite to nano-selenium is a low-cost, green, and efficient method for remediating selenium-polluted wastewater.

[0003] *Sphingopyxis*, belonging to the family Sphingomonadaceae, is widely distributed in soil, water, and plant rhizosphere. Currently, 22 species in this genus have been officially published. *Sphingopyxis* possesses significant potential for degrading various environmental pollutants, such as microcystins, cyhalofop-butyl, and chlorpyrifos, and also has the ability to adsorb cadmium and manganese from water, making it an important microbial resource for bioremediation. [1] There are currently no reports of sphingosine box bacteria biotransforming selenium compounds. Summary of the Invention

[0004] The purpose of this invention is to provide a sphingopyxis sp. R3-92 strain capable of efficiently converting selenite and / or selenate into nano-selenium and its applications.

[0005] To achieve the objectives of this invention, in a first aspect, this invention provides a strain R3-92, isolated and purified from selenium-rich soil in Yutangba, Enshi, Hubei Province, capable of reducing selenite to generate nano-selenium, classified and named Sphingopyxis sp., which is now deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences, Postcode 510070, China, Accession No. GDMCC No: 63729, Deposit date: August 11, 2023.

[0006] Secondly, the present invention provides a microbial preparation containing Sphingosine Trichophyton R3-92.

[0007] Thirdly, this invention provides the application of Sphingosine Box Bacterium R3-92 in the remediation of selenium-contaminated environments and the preparation of nano-selenium.

[0008] Fourthly, the present invention provides a method for the biosynthesis of nano-selenium, wherein Sphingosine Box Bacteria R3-92 is inoculated into a culture medium containing selenite, 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, the initial selenium concentration is 2-80 mM, preferably 2-20 mM, and more preferably 5 mM.

[0010] In one specific embodiment of the present invention, the basic components of the culture medium are: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, solid culture medium with added agar 18 g / L, pH 7.0-7.2.

[0011] Preferably, the culture conditions are: 28±0.5℃ and 150 rpm.

[0012] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0013] The *Sphingosine Tetracycline* R3-92 strain of this invention exhibits strong tolerance to selenate and selenite. Plate tests show that R3-92 can tolerate 100 mM selenate and 80 mM selenite. This invention also establishes a method for the large-scale preparation of nano-selenium using R3-92 in a bioreactor, which can be used for the bioremediation of selenium-containing wastewater and the synthesis of bio-nano-selenium, showing broad application prospects. Attached Figure Description

[0014] Figure 1 The colony and cell morphology characteristics of strain R3-92 of this invention are shown.

[0015] Figure 2This is a phylogenetic tree of the 16S rRNA gene of strain R3-92 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.

[0016] Figure 3 This is a phylogenetic tree of strain R3-92 of this invention, constructed using UBCG based on 92 core genes. Note: Bootstrap analysis was performed using 100 replicates. Bootstrap values ​​(>50%) are labeled at branch points. The numbers in parentheses are the GenBank accession numbers for the strain's genome, and the scale bar represents a 10% nucleotide substitution rate.

[0017] Figure 4 The dDDH and ANI values ​​are for strain R3-92 of this invention and other type strains of the genus *Sphingosine Box*. Note: Strain: 1, R3-92 T ; 2, Sphingopyxis alaskensis RB2256 T ; 3, Sphingopyxisbauzanensis DSM 22271 T ; 4, Sphingopyxis chilensis S37 T 5, Sphingopyxis flavaR11H T ; 6, Sphingopyxis fribergensis Kp5.2 T ; 7, Sphingopyxis granuli NBRC100800 T ; 8, Sphingopyxis indica DS15 T ; 9, Sphingopyxis italica DSM 25229 T ; 10,Sphingopyxis jiangsuensis XHP0097 T ; 11, Sphingopyxis kveilinsis TUF1 T ; 12,Sphingopyxis lindanitolerans WS5A3p T ; 13, Sphingopyxis macrogoltabida 203 T ;14, Sphingopyxis panaciterrae DSM 27164 T; 15, Sphingopyxis panaciterrulae DSM27163 T ; 16, Sphingopyxis soli BL03 T ; 17, Sphingopyxis solisilvae R366 T ; 18,Sphingopyxis terrae NBRC 15098 T ; 19, Sphingopyxis ummariensis UI2 T ; 20,Sphingopyxis witflariensis DSM 14551 T ; 21, Novosphingobium naphthalenivoransNBRC 102051 T .

[0018] Figure 5 The tolerance of strain R3-92 to selenate and selenite in a preferred embodiment of the present invention.

[0019] Figure 6 The reduction ability of strain R3-92 for selenite in a preferred embodiment of the present invention.

[0020] Figure 7 Physicochemical characteristics of the synthesis of nano-selenium by reducing Se(IV) by strain R3-92 of this invention. Detailed Implementation

[0021] 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.

[0022] Example 1: Isolation and Identification of Sphingosine Box Bacteria R3-92

[0023] 1. Strains Isolation

[0024] Five g of rhizosphere soil was collected from selenium-rich soil and added to 95 mL of sterile physiological saline. The mixture was cultured at 25°C and 150 rpm for 30 min with shaking. After a 10-fold serial dilution, 100 μL of the diluted solution was 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°C for 15 min). The plates were cultured at 28°C for 2 days to obtain selenite-tolerant and reducing strains. From these strains, a strain R3-92 capable of reducing selenite to generate nano-selenium was isolated and purified.

[0025] 2. Analysis of colony and cell morphology characteristics

[0026] After incubating for 2 days on TSA agar 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), R3-92 colonies were round with neat edges, light yellow in color, moist on the surface, and did not produce pigment. The colony diameter was 1.0-2.0 mm. Figure 1 a). Observation of R3-92 cell morphology using a Hitachi 7650B transmission electron microscope (TEM) Figure 1 (b) R3-92 cells are short rod-shaped, 1.0-2.0 μm long and 0.6-0.8 μm wide.

[0027] 3. Phylogenetic analysis of the 16S rRNA gene of strain R3-92

[0028] Genomic DNA was extracted from strain R3-92 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℃ -10 min; 94℃ 40 s, 56℃ 40 s, 72℃ 40 s, 30 cycles; 72℃ 10 min. The molecular size of the amplified products was identified by 1% agarose gel electrophoresis. The PCR products were purified and sequenced, and the sequencing results were assembled using DNAMAN software. The 16S rRNA sequence of R3-92 was compared with bacterial 16S rRNA gene sequences 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 R3-92 had the highest similarity with *Sphingopyxis panaciterrae* strains, with R3-92 showing the highest similarity with *Sphingopyxis panaciterrae* Gsoil 124. T The similarity was highest, reaching 99%. The phylogenetic tree of the 16S rRNA gene of strain R3-92, constructed using the neighbor-joining method (Kimura 2-parameter model) with Mega software, is shown below. Figure 2 The display shows R3-92 with Sphingopyxis panaciterrae Gsoil 124 T Gathered in one group.

[0029] 4. Genome sequencing and phylogenetic analysis

[0030] Whole-genome sequencing of strain R3-92 was performed using the Illumina NovaSeq PE150 platform. The results showed that the genome size of R3-92 was 4.8 Mb, with a GC content of 63.3 mol%. Both the genome size and GC content of strain R3-92 were within the range of the *Sphingopyxis* genus (Table 1). A phylogenetic tree of 92 core genes from R3-92 and related taxa of the *Sphingopyxis* genus was constructed using the Universal Bacterial Core Genes (UBCG). Phylogenetic analysis indicated that R3-92 belongs to the *Sphingopyxis* genus and is related to *Sphingopyxis witflariensis* DSM 14551. TClosest kinship ( Figure 3 ).

[0031] Table 1. Basic genomic information of R3-92 and other type strains of the genus *Sphingosine Box*.

[0032] strain Genome size (Mb) G+C (mol%) Genbank login ID R3-92 4.8 63.6 <![CDATA[Sphingopyxis alaskensis RB2256 T ]]> 3.4 65.5 CP000356 <![CDATA[Sphingopyxis bauzanensis DSM 22271 T ]]> 4.3 63.3 NISK00000000 <![CDATA[Sphingopyxis chilensis S37 T ]]> 4.0 65.3 CP142394 <![CDATA[Sphingopyxis flava R11H T ]]> 4.2 63.8 FUYP00000000 <![CDATA[Sphingopyxis fribergensis Kp5.2 T ]]> 5.2 63.8 CP009122 <![CDATA[Sphingopyxis granuli NBRC 100800 T ]]> 4.3 66.4 BCUA00000000 <![CDATA[Sphingopyxis indica DS15 T ]]> 4.1 65.7 FZPA00000000 <![CDATA[Sphingopyxis italica DSM 25229 T ]]> 4.0 65.4 JAATIT000000000 <![CDATA[Sphingopyxis jiangsuensis XHP0097 T ]]> 3.1 64.7 JAILXK000000000 <![CDATA[Sphingopyxis lindanitolerans WS5A3p T ]]> 4.4 65.1 PHFW00000000 <![CDATA[Sphingopyxis lutea DHUNG17 T ]]> 3.1 64.8 JAERPO000000000 <![CDATA[Sphingopyxis macrogoltabida 203 T ]]> 5.7 64.9 CP009429 <![CDATA[Sphingopyxis panaciterrae DSM 27164 T ]]> 4.8 64.8 JAASQN000000000 <![CDATA[Sphingopyxis panaciterrulae DSM 27163 T ]]> 4.4 66.4 JACIJH000000000 <![CDATA[Sphingopyxis soli BL03 T ]]> 3.6 65.8 JAFMTR000000000 <![CDATA[Sphingopyxis solisilvae R366 T ]]> 3.4 64.8 JADKYM000000000 <![CDATA[Sphingopyxis terrae NBRC 15098 T ]]> 4.1 65.0 BCZQ00000000 <![CDATA[Sphingopyxis ummariensis UI2 T ]]> 3.6 65.2 FXWL00000000 <![CDATA[Sphingopyxis witflariensis DSM 14551 T ]]> 4.3 63.4 NISJ00000000 <![CDATA[Novosphingobium naphthalenivorans NBRC 102051 T ]]> 5.2 63.8 BCTX00000000

[0033] 5. Genome similarity analysis

[0034] The average nucleotide similarity (ANI) between its genome and other type strains of *Sphingosine Box* was calculated using JSpeciesWS (https: / / jspecies.ribohost.com / jspeciesws / #analyse). The digital DNA-DNA hybridization (dDDH) value was calculated using GGDC Calculator 3.0 (http: / / ggdc.dsmz.de). Figure 4 The results showed that R3-92 had the highest genomic similarity to *Sphingopyxis witflariensis*, particularly to *Sphingopyxis witflariensis* DSM 14551. T The highest ANI and dDDH values ​​were observed, at 87.0% and 35.4%, respectively; followed by *Sphingopyxis panaciterrae* DSM 27164. T The percentages were 80.8% and 25.4%, respectively. However, the ANI and dDDH values ​​of R3-92 and the type strain of the genus Sphingopyxis were both lower than the species threshold (ANI < 95%, dDDH < 70%), indicating that strain R3-92 is a new species of the genus Sphingopyxis, and it is proposed to be named Sphingopyxis selenitireducens.

[0035] 6. Tolerance test of strain R3-92

[0036] Fresh single colonies of strain R3-92 were inoculated onto TSA agar plates and incubated for 4 days at 4, 10, 20, 28, 30, 32, 34, 37, 40, and 42°C, respectively, to observe the growth of the strain. Single colonies of R3-92 were then inoculated into TSB agar 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°C for 15 min) and activated by shaking at 28°C and 150 rpm for 12 h. OD was then adjusted. 600The pH was set at 0.8 as the seed culture. 1% R3-92 seed culture was inoculated into TSA medium containing 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8% (w / v) NaCl, respectively, and incubated at 28℃ and 150 rpm for 4 days. Growth was observed. The pH of TSB medium was adjusted to 4, 5, 6, 7, 8, 9, 10, and 11 with citric acid (0.1 M) and NaOH (0.1 M), respectively. 1% R3-92 seed culture was then inoculated into the medium, and incubated at 28℃ and 150 rpm for 4 days. Growth was observed. The results showed that strain R3-92 had higher tolerance to temperature and salinity than its closely related relatives, with a growth temperature range of 4-37℃ and a growth pH range of 7-10. It could tolerate 4% NaCl (Table 2).

[0037] 7. Measurement of physiological and biochemical indicators

[0038] According to the "Handbook for Identifying Common Bacteria" [2] The physiological and biochemical characteristics of strain R3-92 were determined using conventional microbial identification methods. The physiological and biochemical characteristics of R3-92 and the reference strain are shown in Table 2. Microbiological characteristics and physiological and biochemical properties of strain R3-92: Gram-negative; after culturing in TSA medium at 28℃ for 2 days, colonies are 1-2 mm in diameter, yellow, do not produce pigment, have a moist and glossy surface, and neat edges; aerobic growth; growth temperature range 4-37℃, growth pH range 7-10, and can tolerate 4% NaCl. Negative for catalase and oxidase. Capable of reducing nitrates and hydrolyzing esculin, but not gelatin. Does not synthesize indole, and does not produce arginine dihydrolase, urease, or β-galactosidase. It does not ferment D-glucose and can utilize glucose and N-acetylglucosamine, but it cannot utilize carbon sources such as L-arabinose, D-mannose, D-mannitol, D-maltose, gluconic acid, decanoic acid, adipic acid, malic acid, citric acid, or phenylacetic acid.

[0039] Table 2 Physiological and biochemical indicators of R3-92 and reference strains

[0040] index R3-92 <![CDATA[ Sphingopyxis witflariensis W-50 T ]]> <![CDATA[ Sphingopyxis panaciterrae Gsoil 124 T <!-- 4 -->]]> Temperature tolerance (°C) 4-37 15-37 10-30 pH tolerance 7-10 ND 5.5-8.5 NaCl tolerance (%, w / v) 0-4 ND 0-3 Oxidase - + + catalase + + + Nitrate reduction + - - D-glucose utilization + + + Arginine dihydrolase - - - Urease - - -

[0041] Note: +, positive; -, negative; ND, no data.

[0042] Based on the phylogenetic analysis, genome sequence similarity comparison, and the aforementioned microbiological characteristics and physiological and biochemical properties, strain R3-92 was identified as a new species of the genus *Sphingosine Box*. This strain has been deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 11, 2023, with accession number GDMCC No. 63729.

[0043] Example 2: Tolerance of strain R3-92 to selenate and selenite

[0044] Single colonies of R3-92 were picked and inoculated into LB tubes (yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, solid medium supplemented with agar 18 g / L, pH 7.0-7.2, sterilized at 121℃ for 20 min), and activated by shaking at 28℃ and 150 rpm for 12 h. OD was then adjusted. 600 The concentration was 0.8, which was used as the seed culture. The R3-92 seed culture was serially diluted with sterile physiological saline in a 10-fold gradient, and 2.5 μL of each solution was added to a selenium-containing plate. After being dried with sterile air, the plate was incubated at 28°C for 48 h.

[0045] The growth of strain R3-92 on selenium-containing plates is shown in the figure. Figure 5 The results showed that R3-92 could tolerate 100 mM selenate (sodium selenate) and 80 mM selenite (sodium selenite). Colonies grown on selenite plates were bright red, indicating that R3-92 could reduce selenite to red selenium nanoparticles. On plates with high concentrations of selenate, R3-92 colonies showed a lighter red color, indicating that it could generate small amounts of selenium nanoparticles under high-concentration selenate conditions.

[0046] Example 3: The reducing ability of strain R3-92 to selenite

[0047] Inoculate 0.5 mL of R3-92 seed culture (OD) into a 100 mL shake flask containing 50 mL of LB medium. 600 =0.8) and the corresponding volume of sodium selenite (filtered and sterilized) to make initial selenium concentrations of 2 mM, 5 mM, 10 mM and 20 mM (as Se), respectively, and incubated at 28℃ and 150 rpm for 48 h with shaking.

[0048] Using sodium sulfide spectrophotometry [3] Determination of nano-selenium yield: Centrifuge 500 μL of the test bacterial solution at 12000 rpm for 5 min, wash three times with sterile physiological saline, discard the supernatant, 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. Results are shown below. Figure 6 Under treatment with 2-20 mM Se(IV), strain R3-92 was able to synthesize selenium nanoparticles, and the yield of selenium nanoparticles gradually increased within the range of 0.31-0.67 mM. The highest conversion rate of selenium nanoparticles, reaching 15.5%, was observed at a Se(IV) concentration of 2 mM.

[0049] Example 4 Physicochemical characteristics of selenium nanoparticles synthesized by strain R3-92

[0050] R3-92 seed liquid (OD) 600 =0.8) was inoculated at 1% v / v in LB medium containing 5 mM sodium selenite and cultured at 28℃ and 150 rpm for 48 h with shaking. The physicochemical characteristics of the nano-selenium synthesized by strain R3-92, such as shape, particle size and element type, were analyzed by transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS).

[0051] The results showed that the selenium nanoparticles synthesized by strain R3-92 through the reduction of Se(IV) were spherical with a particle size between 100 and 300 nm, and mainly attached to the extracellular matrix of bacteria. Figure 7 EDS analysis of the nanoparticles showed characteristic peaks of selenium at 1.37 keV, 11.22 keV, and 12.50 keV, with a selenium mass ratio of 97.0%, indicating that the nanoparticles synthesized by the strain were nano-selenium.

[0052] Example 5: Efficiency of strain R3-92 in synthesizing nano-selenium in a bioreactor

[0053] Add 6 L of fermentation medium (20 g / L glucose, 10 g / L yeast extract, 10 g / L soybean peptone, 5 g / L sodium chloride, 2 g / L ammonium sulfate, 1 g / L ammonium chloride, 0.3 g / L calcium chloride, 0.5 g / L potassium dihydrogen phosphate, 0.3 g / L magnesium sulfate, pH 7.0-7.2) to a 10 L fermenter, sterilize at 121℃ for 20 min, and after the medium has cooled, inoculate with 1% v / v R3-92 seed culture (OD200). 600 =0.8), and 30 mL of sterile 1 M Se(IV) mother liquor (sodium selenite) was added to make the initial selenium concentration 5 mM. The temperature was 28 ± 0.5℃, the rotation speed was 150 rpm, and the aeration rate was 0.5 vvm throughout the fermentation process. After 24 h of fermentation, the content of nano-selenium in the fermentation broth was determined by sodium sulfide spectrophotometry. The results showed that after 24 h of fermentation, strain R3-92 could reduce and generate 0.83 mM nano-selenium, with an 84% increase in yield and a 50% reduction in fermentation time.

[0054] Example 6: Isolation and purification of nano-selenium synthesized by strain R3-92

[0055] 1. Separation and Purification: The fermentation broth prepared in Example 5 was centrifuged at 8000 rpm for 10 min. The resulting red precipitate was washed three times with sterile physiological saline and resuspended in purified water to obtain a nano-selenium suspension. The nano-selenium suspension was transferred to an extraction tower, 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 to obtain bio-nano-selenium.

[0056] 2. Freeze-drying:

[0057] The prepared bio-nano selenium was frozen in liquid nitrogen for 10-15 min and then freeze-dried in a freeze dryer. The freeze-drying parameters were: pressure 20-100 Pa, heating plate temperature 20-35℃, and sample thickness 10-25 mm. The drying time was 48-72 h to obtain bio-nano selenium dry powder A.

[0058] The prepared bio-nano selenium was frozen in liquid nitrogen for 10-15 min and then freeze-dried in a freeze dryer. The freeze-drying parameters were: pressure 20-65 Pa, heating plate temperature 20-25℃, and sample thickness 10-14 mm. The drying time was 36-48 h to obtain pure bio-nano selenium powder B.

[0059] Example 7: Application of bio-nano selenium in selenium-enriched fertilizers, feeds, functional foods, health products, and pharmaceuticals

[0060] Example 1: Bio-nano selenium powders A and B were suspended in purified water to prepare selenium-enriched fertilizers A and B at concentrations of 1-5 g / L. Fertilizers A and B were then used in the cultivation of grain crops such as wheat, rice, and corn; miscellaneous grains such as soybeans, peanuts, millet, and sweet potatoes; edible fungi such as enoki mushrooms, shiitake mushrooms, and wood ear mushrooms; vegetables such as tomatoes, eggplants, and cucumbers; and fruits such as apples and kiwis, as well as tea cultivation, to obtain reprocessable selenium-enriched crops, selenium-enriched edible fungi, selenium-enriched fruits, and selenium-enriched tea. The selenium content of selenium-enriched grains and miscellaneous grains was 100-300 μg / kg, the selenium content of selenium-enriched vegetables and fruits was 20-100 μg / kg, and the selenium content of selenium-enriched edible fungi was 150-5000 μg / kg.

[0061] Example 2: Mix bio-nano selenium powder A or B with feed ingredients at a ratio of 50-800 μg / kg to prepare selenium-enriched feed A and selenium-enriched feed B. Feed A and B are then fed to laying hens, broilers, pigs, sheep, cattle, and other livestock and poultry to obtain selenium-enriched eggs, selenium-enriched chicken, selenium-enriched pork, selenium-enriched mutton, and selenium-enriched beef that can be further processed.

[0062] Example 3: Bio-nano selenium powder B is mixed evenly with millet flour, vegetable oil, and purified water (weight percentages of 55%, 10%, and 35%, respectively) at a ratio of 10-2500 μg / kg. The mixture is then extruded and puffed in an extruder, dried, and packaged to obtain puffed millet selenium-enriched functional food. Alternatively, replacing the millet flour with corn flour, buckwheat flour, or soybean flour yields puffed corn, buckwheat, or soybean flour selenium-enriched functional food.

[0063] Example 4: Mix bio-nano selenium dry powder B (10-500 mg / kg) with starch (g / kg), vitamin E (0-22 g / kg) and β-carotene (0-5 g / kg) evenly, add a wetting agent, and make microparticles in a granulator. Dry the microparticles and fill them into capsule shells, controlling the weight of each capsule to 0.3-0.6 g. Bottle 100 capsules per bottle, seal and store.

[0064] Example 5: Mix bio-nano selenium dry powder B (50-800 mg / kg) with starch and plant protein powder (weight ratio of starch to plant protein powder is 95:4.9) evenly, add the binder HPMC to the above mixture, stir evenly in a mixer, put the raw materials into a tablet press and start the tablet press, dry, each tablet weighs 0.4-0.6 g, bottle 100 tablets per bottle, seal and store.

[0065] 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.

[0066] References:

[0067] [1]Sharma M, Khurana H, Singh DN, Negi R K. The genus Sphingopyxis: Systematics, ecology, and bioremediation potential - A review [J]. Journal of Environmental Management, 2021, 280.

[0068] [2] Dong Xiuzhu, Cai Miaoying. Handbook of Systematic Identification of Common Bacteria [M]. Handbook of Systematic Identification of Common Bacteria, 2001.

[0069] [3]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.

Claims

1. Sphingopyxis sp. R3-92, accession number GDMCC No: 63729.

2. A microbial preparation containing the Sphingosine Box bacteria of claim 1.

3. The application of the Sphingosine Box bacteria described in claim 1 in the remediation of selenium-contaminated environments and the preparation of nano-selenium.

4. A method for the biosynthesis of nano-selenium, characterized in that, The sphingosine box bacteria of claim 1 were 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, The initial selenium concentration in the culture medium containing selenite is 2-80 mM.

6. The method according to claim 5, characterized in that, The initial selenite concentration in the culture medium containing selenite is 2-20 mM.

7. The method according to claim 6, characterized in that, The initial selenium concentration in the culture medium containing selenite was 5 mM.

8. The method according to any one of claims 4-7, characterized in that, The basic components of the culture medium are: yeast extract 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, solid culture medium with added agar 18 g / L, pH 7.0-7.

2.

9. The method according to any one of claims 4-7, characterized in that, The cultivation conditions were: 28±0.5℃ and 150 rpm.

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