Pseudomonas sp. strain as well as cultivation method and application thereof
A Pseudomonas, enrichment medium technology, applied in the direction of chemical instruments and methods, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of bacteria that have not yet degraded phenol, and achieve high-efficiency phenol degradation performance , less cumulative effect
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Embodiment 1
[0030] Embodiment 1: Screening and performance determination of Pseudomonas CGMCC No.13433
[0031] The activated sludge is taken to a sewage treatment tank of a factory, and 5mL of mud-water mixture is taken from the activated sludge into 45mL of sterilized heterotrophic nitrification medium. The formula of heterotrophic nitrification medium is: (NH 4 ) 2 SO 4 0.945g / L, sodium citrate 6.536g / L, MgSO 4 ·7H 2 O 1g / L, NaCl 0.12g / L, MnSO 4 ·H 2 O 0.01g / L, FeSO 4 ·7H 2 O 0.02g / L, KH 2 PO 4 0.2g / L, Na 2 HPO 4 0.3g / L, pH 7.0-7.5. Then placed at 30°C, 200rpm air bath shaker enrichment culture for 12h. After gradient dilution of the enriched solution, spread evenly on the heterotrophic nitrification solid medium (agar 20g / L, the rest of the components are the same as the heterotrophic nitrification medium). After culturing in a constant temperature incubator at 30°C for 1 day, single clones with different shapes and sizes were picked, streaked and purified, and number...
Embodiment 2
[0035] Embodiment 2: Molecular biology identification of bacterial strain
[0036] The strains were identified by 16S rDNA sequence alignment. The total DNA of Pseudomonas CGMCC No.13433 was extracted, and the strain 16S rDNA was amplified by a pair of universal primers. The upstream primer was 8f (5'-AGAGTTTGATCCTGGCTCA-3'), and the downstream primer was 1492r (5'-GGTTACCTTGTTACGACTT-3'). PCR reaction system (50 μL): template DNA 1 μL, PCR Taqmix 25 μL, upstream and downstream primers 1.5 μL, DMSO 2 μL, add ddH 2 O to 50 μL of the reaction system. PCR program: 94°C for 5min, 30 cycles of 94°C for 30s, 55°C for 30s, 72°C for 1min and 30s, 72°C for 10min, 4°C for 5min. PCR products were purified and sequenced by Meiji Biomedical Technology Co., Ltd. The 16S rDNA sequence obtained by sequencing was submitted to NCBI, and the homology sequence comparison analysis was carried out through the software Blast and GenBank, and the phylogenetic tree of the strain was constructed us...
Embodiment 3
[0040] Embodiment 3: the resistance research of bacterial strain
[0041] Prepare LB plate with common resistance, LB medium formula: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, agar 20g / L. Antibiotics were selected for common resistance, including kan, ampicillin (amp), chloramphenicol (cm), streptomycin (sm) and tetracycline (tc), and antibiotics were added to LB medium at a ratio of 1:1000. Pick the strains and smear them on the resistant plates, and culture them in a 30°C incubator. After 1 day, the growth of the strain was observed. As shown in Table 1, Pseudomonas CGMCC No.13433 was resistant to amp, cm, sm and tc, but not to kan.
[0042] Table 1 The resistance result table of Pseudomonas CGMCC No.13433
[0043] can
[0044] In the table, "+" means positive; "-" means negative.
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