A strain of Bacillus mohaiwei and its application
A technology of Bacillus and Bacillus, applied in the field of environmental microbial applications, to achieve the effects of high degradation rate, simple operation, and short degradation cycle
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Embodiment 1
[0034] Identification of Example 1 bacterial strain B1811
[0035] Strain B1811 was isolated from soil, and its morphology was as follows: figure 1 As shown, it is a rod-shaped Gram-negative bacteria that can form spores. Genomic DNA was extracted, and the genomic DNA was subjected to polymerase chain reaction (Polymerase Chain Reaction, PCR) using 16s rDNA and BCR1 primers to propagate specific DNA sequences, and the National Center for Biotechnology Information (National Center for Biotechnology Information, NCBI) database for strain comparison.
[0036] (1) 16S rDNA sequence analysis:
[0037] 16s rDNA forward primer 27F: 5'-AGAGTT TGATCC TGG CTC AG-3', as shown in SEQ ID NO.1;
[0038] 16s rDNA reverse primer 1541R: 5'-AAG GAG GTG ATC CAG CC-3', as shown in SEQ ID NO.2; the amplified 16S rDNA sequence is shown in SEQ ID NO:3, and the full length of the sequence is 1461bp. The amplified 16S rDNA sequence was compared with the gene sequence of related strains in the GenB...
Embodiment 2
[0044] Embodiment 2 Preparation of Bacillus mohaiwei B1811 liquid shake flask culture solution
[0045] (1) Slant culture: Bacillus mohaiwei B1811 was inoculated on the slant medium and cultured at 40° C. for 48 hours to obtain the slant strain. The slant medium used contained the following ingredients per 1L: 2.0g glucose, 1.0g peptone, 0.5g yeast extract, 1.8g agar, the rest of distilled water, neutral pH, and sterilized at 115°C for 20min.
[0046] (2) The slant strain was inoculated into the sterilized seed culture medium, and cultured with constant temperature shaking at 175 rpm and 30° C. for 24 hours to obtain a seed culture solution. The seed culture medium contains the following ingredients per 1L: 0.5g of ammonium sulfate, 4.0g of sodium chloride, 0.5g of potassium phosphate trihydrate, 0.4g of magnesium sulfate heptahydrate, 15g of agar powder, 5g of yeast extract, and the remainder of distilled water. Quantity, pH7.0, sterilized at 121°C for 25min.
[0047] (3) A...
Embodiment 3D
[0048] Example 3DBP standard curve formulation and assay
[0049] (1) High-performance liquid chromatography (HPLC) to prepare a standard curve: take 600 μL of DBP and use methanol as a solvent to prepare a 1 mg / mL DBP solution. After diluting ten times, take 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, Dilute 1.0mL, 1.2mL, and 1.4mL into 2mL centrifuge tubes, that is, the concentration gradient (μg / mL) is 10, 20, 30, 40, 50, 60, and 70, respectively, and filter them with a 0.45μm organic filter membrane and place in the liquid Phase vials to be tested. The HPLC detection conditions in this embodiment 3 are: the chromatographic column is a Sepax Gp-C18 column (150mm*4.6mm, 5.0 μm); the mobile phase is acetonitrile-water (83:17, V / V); the injection volume 10μL; flow rate 1.0mL / min; column temperature 25C°; UV detector wavelength 210nm. Take the peak area of DBP as the abscissa and the concentration of DBP as the ordinate to make a standard curve; then obtain the peak area-concentration ...
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