Para-chloro nitrobenzene degrading testosterone coma monad and its use
A technology of Comamonas, chlorinated nitrobenzene, applied in the field of microbial biotechnology and environmental biology
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Embodiment 1
[0016] Example 1: Culture and biological characteristics of Comamonas testosteroni CNB1 CGMCC No.1028.
[0017] Comamonas testosteroni CNB1 CGMCC No.1028 was inoculated in inorganic salt medium, the medium composition: disodium hydrogen phosphate 1g, potassium dihydrogen phosphate 0.5g, MgSO 4 ·7H 2 O 0.03g, trace element solution 5mL, p-chloronitrobenzene 200mg, distilled water 1000mL, pH7.0, add 15g / L agar to the solid medium. Sterilize at 121°C for 30 minutes. Shake the culture on a shaker at 28-30°C and 130rpm. The bacterium has the following characteristics: (1) The size of the colony cultivated on the LB plate for 2 days is 1-2 mm in diameter, and the colony is round, with a smooth surface, neat edges, raised, and colorless. (2) Cell morphology characteristics of Comamonas testosteroni (Comamonastestosteroni) CNB1 CGMCC No.1028: suitable for normal temperature culture in the range of pH 6-11, the cells are short rod-shaped, with 2 extreme hairs, Gram staining negative...
Embodiment 2
[0024] Embodiment 2: PCR amplification and sequence determination of the 16S rRNA gene of Comamonas testosteroni CNB1 CGMCC No.1028
[0025] Comamonas testosteroni CNB1 CGMCC No.1028 was inoculated on the LB plate, directly picked a ring of bacteria from the plate, added 100 μL sterile redistilled water, vortex mixed, boiled water bath for 2 minutes, centrifuged at 12000r / min for 5 minutes, and the supernatant was directly for PCR. The primers used for the PCR reaction of 16SrDNA amplification are a pair of universal primers. Forward primer Pf: 5'-AGAGTTTGATCCTGGCTCAG-3'; reverse primer Pr: 5'-ACGGCTACCTTGTTACGACT-3', corresponding to 8-27 and 1495-1514 bases of the 16S rRNA gene of Escherichia coli, respectively. The PCR reaction system (50μL) is: 10×buffer 5μL, 25mmol / L MgCl 2 4 μL, 10 mmol / L dNTPs 1 μL, 30 pmol / L primers 1 μL, BSA 0.5 μL, ddH 2 0.37 μL, TaqDNase 0.4 μL. The PCR reaction conditions were: 95°C for 4min, 95°C for 1min, 48°C for 1min, 72°C for 1min, 30 cyc...
Embodiment 3
[0026] Embodiment 3: Comamonas testosteroni CNB1 CGMCC No.1028 provided by the present invention is to the degradation of p-chloronitrobenzene
[0027] Comamonas testosteroni CNB1CGMCCNo.1028 strain culture fluid (OD 600 =0.2) Inoculate in the culture medium that contains p-chloronitrobenzene (20mg / L) by 1% of the inoculation amount, at 30 DEG C, shake culture on the shaker of 130r / min, regularly take samples to measure thalline growth, p-chlor Nitrobenzene content and chloride ion concentration, the results are as follows figure 1 shown. figure 1 It shows that the growth of CNB1 bacteria and the degradation of p-chloronitrobenzene have a short lag period, and then enter the logarithmic phase, and reach the highest growth amount in the 20th hour, and the degradation rate reaches 87% at this time, and the p-chloronitrobenzene The decrease in moles of benzene is comparable to the increase in moles of chloride ions.
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