| As the most widely usedtetracycline antibiotics,oxytetracycline cause a large amount of residues in the environment,which poses a great threat to life health and environmental ecological security.Therefore,it is of great significance to explore efficient and environmentally friendly technical measures for ectopic treatment of oxytetracycline-contaminated wastewater and waste and to remediate oxytetracycline-polluted environment.Consideringthe advantages of microbial technology in the treatment and remediation of organic pollutants,and the issue of biosafety,Arthrobacter nicotianae OTC-16,a high-efficiency oxytetracycline degrading bacterium obtained in the previous experiments,was used to study the removal effect of the strain OTC-16 on soil oxytetracycline in the presence of rhizosphere pathogenic fungi and pesticides;The biodegradable metabolites of oxytetracycline were identified and the ecotoxicity was evaluated;finally,the expression of five antibiotic resistance genes on the chromosomes and plasmids of the degrading bacterium OTC-16 in the process of oxytetracycline degradation was explored,and the change of the abundance of the antibiotic resistance genes in the soil were analyzed by using q PCR.The results are as follows:(1)The degradation effect and response mechanism of oxytetracycline efficient degradation bacterium OTC-16 in facing pathogenic fungi and pesticide in cucumber rhizosphere soil were tested by the paper-dish method and pot experiments.The results showed that strain OTC-16 inducing into the rhizosphere of cucumber could effectively degrade oxytetracycline(OTC)and accelerate the decomposition of OTC.The strain had a wide range of environmental adaptability.However,some biotic factor,such asplant pathogen Fusarium oxysporum,and abiotic factor such as fungicide thiram in the study may affect strain's degradation effect by inhibiting the growth of degrading strains.(2)Bacterial strain Escherichia Coli and Bacillus subtilis,as well as alga Scenedesmus obliquus were used as indicators to evaluate the ecotoxicity of the oxytetracycline degradation products.The results showed that the growth of B.subtilis and E.coli in the treatment solution of oxytetracycline after degradeby OTC-16 was significantly better than that of negative control and abiotic treatment,and no significant difference was observed when compared with the positive control,which contained no oxytetracycline.The results confirmed that the biotoxicity of oxytetracycline metabolites degraded by OTC-16 strain was greatly reduced compared with the original medicine.The results of toxicity of oxytetracycline degradation products to S.obliquuswas consistent with bacterial test.The growth of S.obliquus in oxytetracycline solution treated with OTC-16 degradation strain was significantly better than the natural hydrolyzed oxytetracycline treatment and negative control.However,compared with the positive control,the chlorophyll content of algal cells in oxytetracycline treated with OTC-16 strain was significantly reduced,which means strain OTC-16 did biotransform OTC into low-toxicity products,but there was still certain negative effect on chlorophyll synthesis.Observing the ultrastructure of S.obliquus under transmission electron microscope,found that the structure of S.obliquus in the solution of oxytetracycline degraded by degrading bacterium OTC-16 was better than naturally hydrolyzed and negative control in which the chloroplasts and cell walls of S.obliquus was obviously damaged.The results proved that the degrading strain OTC-16 can biotransform oxytetracycline,and played an role in reducing the ecological toxicity of oxytetracycline.The degradation products of oxytetracycline were detected by HPLC-Q-TOF-MS,and finally,four degradation byproducts were identified,including EOTC,ISO-OTC,demethylation OTC(TP447),and aminobenzoic acid(TP138).It was speculated that the reduction of biotoxicity ofthe degradation products after the addition of OTC-16 may be related to the decrease of accumulation of oxytetracycline's toxic hydrolytic products such as EOTC.(3)Five antibiotic resistance genes tet V,sul1,tet33,ant2 ia,cml?e8 were found in the chromosome and plasmids of strain OTC-16 based on the whole genome sequencing.Real-time quantitative PCR(RT-q PCR)was used to track the expression of 5 resistance genes in the OTC degradation process by strain OTC-16.The results showed that the addition of oxytetracycline obviously promoted the expression of gene ant2 ia,sul1 and tet33 in the early experiments,but did not affect or inhibit the expression of tet V and cml?e8.Analyzing the location of the five resistance genes of strain OTC-16 indicated that the plasmid-derived resistance genes have high expression activity.Meanwhile,the results of soil experiments showed that the abundance of three genes,tet33?ant2ia and sul1 on plasmid 2,significantly increased 10 d later after strain OTC-16 was induced into soil.From the high transcriptional activity and the abundance of resistance genes in soil,it could be inferred that the genes tet33?ant2ia and sul1 might bring high risks for environment,and some gene manipulation technologies,such as removing resistance plasmid and knockouting resistance genes in degrading strain OTC-16 would be effective ways to reduce some of the security risks. |
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