Method for biosynthesizing nano-selenium by Bacillus subtilis and its application
A technology of Bacillus subtilis and nano-selenium, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, applications, etc., can solve the problems of non-factory production and application, and achieve high yield, environmental friendliness, and remarkable effect Effect
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Embodiment 1
[0045] Example 1 Isolation, purification and identification of Bacillus subtilis S12
[0046] 1.1 Isolation and purification of strain S12
[0047] A strain S12, which can tolerate higher concentration of sodium selenite, was isolated from the soil.
[0048] 1.2 Identification of strain S12
[0049] 1.2.1 PCR amplification of 16S rDNA and gyrA gene sequence and sequencing:
[0050] Inoculate strain S12 on LB solid medium and culture for 24h, take 0.2mL sterilized PCR tube, add 10μL ddH 2 O,
[0051] Pick up a single colony with a sterile toothpick and put it into a PCR tube and mix well.
[0052] 1.2.2 Construct the PCR reaction system:
[0053] 16S rDNA: Using 8F (5'-CGGGATCCAGAGTTTGATCCTGGCTCAGAACGAACGCT-3') and 1506R (5'-CGGGATCCTACGGCTACCTTGTTACGACTTCACCCC-3') as primers, the 16S rDNA gene sequence was amplified by PCR. The PCR reaction system is: ddH 2 O, 18.5 μL; 10×Buffer, 2.5 μL, dNTPMix, 2 μL; 8F, 0.5 μL; 1506R, 0.5 μL; bacterial solution, 0.5 μL; rTaq DNA poly...
Embodiment 2
[0058] Embodiment 2 Bacillus subtilis S12 is to the tolerance concentration of sodium selenite
[0059] 2.1 Prepare solid LB with different concentrations of selenium-containing medium (each liter of medium contains 10g of NaCl, 10g of tryptone, 5g of yeast extract, 15g of agar, and 1L of deionized water), autoclave at 121°C for 20min; prepare 1M selenous acid The sodium mother liquor was sterilized by filtration, and sodium selenite solution was added to make the sodium selenite content in the culture medium 0mM, 10mM, 25mM, 35mM, 55mM, 70mM, 80mM respectively.
[0060] 2.2 Pick a single colony of the S12 strain and inoculate it in 5ml LB liquid medium for 8 hours (150rpm, 28°C) and shake it for 8 hours to take the above bacterial solution and dilute it to OD 600 =0.8 of the mother liquor for later use; the mother liquor was diluted to 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 , respectively drop 2.5 μL of bacterial solutions of different concentrations on the selenium-containin...
Embodiment 3
[0062] Embodiment 3 Bacillus subtilis S12 is to the conversion efficiency of sodium selenite
[0063] 3.1 Prepare different concentrations of selenium-containing liquid LB medium, take 5mL and divide it into test tubes, and sterilize under high pressure at 121°C for 20 minutes; prepare 1M sodium selenite mother solution, filter and sterilize, add sodium selenite solution, and make the medium The contents of sodium selenite were 1mM, 3mM, 5mM, 7mM, 10mM, 15mM, 20mM, respectively, and each concentration gradient was repeated three times.
[0064] 3.2 Pick a single colony of the S12 strain and inoculate it in 5ml LB liquid medium for shaking culture for 8 hours (150rpm, 28°C) to take the above bacterial solution and dilute to OD 600 =0.8; the diluted bacterial solution was inoculated in LB medium (containing Na 2 SeO 3 ), shake and cultivate for 48 hours.
[0065] 3.3 Prepare 1M Na with distilled water 2 S solution (prepared and used now); take 1ml of bacteria and nano-seleni...
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