Isolation And Identificationof Oxytetracycline-degrading Strain And Its Use In Bioremediation Simulation Of Contaminated Soil

Oxytetracycline(OTC) is a broad-spectrum antimicrobial drug widely used in aquaculture and livestock. Due to its low cost, OTC is among the most common antibiotics used as an antimicrobial and promoting-growth additives in animal husbandry, resulting in a large number of residues in the environment. The persistent antibiotic residues may lead to the development of antibiotic-resistant bacteria and thus pose a threat to human health. At present, biodegradation with the advantages, such as low cost, thorough degradation and so on, has become the focus of the research on the removal of antibiotic residues at home and abroad. In order to remove the residues of oxytetracycline in the environment, in this paper oxytetracycline-degarading strains were isolated from soil with long-term oxytetracycline pollution using the method of enrichment and acclimation. The strain with highest degradation rate was identified by morphological observation and ITS sequencing, then studying its degradation characteristics in the medium and its degradation behavior in artificially contaminated soil. The results are as follows:(1) In this paper, 9 strains which could grow in the medium with OTC as sole carbon source were obtained using the method of enrichment and acclimation. When the mobile phase was the mixture of oxalic acid(0.01 mol/L), acetonitrile and methanol(76: 16: 8, V/V/V), the recovery of OTC ranged from 78.07% to 94.63% in the medium and the average coefficient of variation ranged from 2.48% to 6.67%, the ability of degrading OTC of the isolated strains were detected by using the high performance liquid chromatography(HPLC), then the strains named Y-1, Y-3and Y-7 which were able to degrade OTC evidently were found, with OTC degradation rate of7.79%, 10.42% and 31.86%, respectively, among which the strain Y-7 had the best ability of degrading OTC. According to the growth of the strain Y-7 in the medium and observing the strain Y-7 by scanning electron microscopy, the strain Y-7 was identified as the fungus. Using PCR amplification of the ribosomal ITS gene region and sequencing, the ITS sequence with the length of 522 bp was obtained. Through the sequence alignment on BLAST, the strain Y-7 was identified as Aspergillus sp. and the max identity reached 100%.(2) The optimum culture condition of the strain Y-7 degrading OTC in the inorganic salt medium was: 40℃, pH6, culture time of 7d, medium volume of 2% and substrate concentrationof 100mg/L. From this we can see that the oxytetracycline-degrading strain Y-7 showed high temperature resistance and the degradation rate of OTC was higher under neutral pH. The degradation rate of OTC in the medium with strain Y-7 rose with the increase of both temperature and substrate concentration, while the degradation rates of OTC showed an upward trend before declining with the rise of all of pH, culture time and medium volume.(3) OTC in soil was extracted using the method of HLB solid phase extraction. The range of the OTC recovery in soil was 63.16%~80.20% and the range of variation coefficient was2.01%~9.53%(<15%), thus this method was accurate for extraction of oxytetracycline in the soil.Compared with OTC concentration in soil without the strain Y-7, the degradation rate of OTC(the initial concentration of 50 mg/kg) in soil with the strain Y-7 was 30.63%, which showed that the strain Y-7 could degrade oxytetracycline in the soil.