| Tetracycline was the essential drug of agricultural production and breeding. And it’s one of the largest drug usage in the world. It will lead to drug residues and pollute the environment if used a large amount for a long time. Tetracyclines will exist in soil, waters, even the bodies of animal and plant in the form of active compound, and access to human body treat to human health through food chain if can’t be handled in time. Facing the problem of pesticide residue increasingly serious, microbial degradation was one of the hot research at home and abroad, because it had some advantages, such as low costs, degradation of thoroughly and good applicability. The test was for the purpose of tetracycline antibiotics, separation and screened strains degrading bacteria in the soil polluted by tetracycline in the long time, and study its degradation characteristics.1. Firstly, the test optimize the method of detection on the HPLC about 3 kinds of tetracycline antibiotics oxytetracycline, tetracycline, chlortetracycline. The experimental results showed that In 0.01 mol·L-1 oxalic acid/acetonitrile/methanol (68/24/8, V/V/V) as mobile phase, column temperature 25 ℃.The flow rate of 1 mL·min-1, the effect of detection is best when sample amount to 20 μL and wavelength of 360 nm, the peak time earlier.2. Tetracycline antibiotics were soluble in water, so choose Na2EDTA-Mcllvaine buffer as extract in the test. Firstly, with high-speed centrifugal method to remove the medium of microorganisms; secondly, using N-hexane and chloroform remove the lipid degradation bacteria in the training process; finally, testing the centrifugal supernatant on detection. The experimental results of adding recovery showed that the recovery of three kinds of tetracycline recovery was between 79%-92%, which meet the test requirements.3. The preliminary screening test 6 strains capable of degrading strains were isolated in from the soil polluted by tetracyclines in the long time. After weeks of repeating domesticated screening, the results showed that six strains degrading bacteria to tetracyclines had a different degree of degradation, and tetracyclines as the sole carbon source. The degradation effect of TJ-2# bacteria was the most significant, followed by TJ-6# bacteria. Through the test of gram staining and other physiological and biochemical indexes and 16S rDNA detection, it confirmed that degradation bacteria TJ-2# was Achromobacter xylosoxidans; and TJ-6# bacteria was Bacillus subtilis.4. In the study that different carbon and nitrogen sources on the degrading bacteria growth and degradation characteristics of TJ-2#, tests set up different processing. The sugar cane was the most significant source of carbon for degrading bacteria growth and degradation effect of the three kinds of tetracyclines. Organic nitrogen source was peptone. Through the record of 7d, we could find that TJ-2# bacteria was the fastest growth in 1-3d, the ability of degradation of three kinds of tetracyclines was strongest; growth was the largest on the 4d, then growth was restrained, leveled off. Degradation rate was maximum in 5d, so as the test of time.5. In the study that temperature and pH effect the degradation bacteria growth and degradation characteristics of TJ-2# we could find that temperature of 35℃ and pH=7 strains was best of growth, and maintaining higher degradation rate about three kinds of tetracyclines. In addition, degradation bacteria TJ-2# had the highest growth and degradation rate when the liquid volume was the 60 mL, inoculation amount was 2%, and the substrate concentration was 60 mg-L-1. |
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