| Residual tetracycline and oxytetracycline,and their induced antibiotic resistance genes?ARGs?have been highly detected in multi-environmental media.In particular,aquaculture wastewater with a high residue of tetracycline and oxytetracycline also have a high concentration of ammonia and nitrate,which will bring about potentially huge ecological and environmental risks.Thus,screening for both,highly efficient tetracycline and oxytetracycline biodegradation,and simultaneously nitrogen conversion?biological denitrification?,is a key technical strategy.A novel tetracycline degrading indigenous strain and an oxytetracycline degrading indigenous strain,were isolated from municipal sludge after long-term domestication and screening.Physiological and biochemical characteristics,biodegradation characteristics,biodegradation kinetics,biodegradation pathway and mechanism,nitrogen conversion and carbon effect,and the treatment efficiency of Moving Bed Biofilm Reactor?MBBR?system for pollutants in simulated aquaculture wastewater were investigated.The main results are as follows:?1?Physiological and biochemical characteristics,Biology analysis,16S rDNA sequencing and napA gene amplification of the degrading bacteria were studied by using biological experiments.Tetracycline-degrading strain SQY5 and oxytetracycline-degrading strain KSS10 were identified as Klebsiella sp.SQY5 and Ochrobactrum sp.KSS10,respectively.The 34 and 14 carbon sources could be utilized by strain SQY5 and KSS10,respectively,and all of them could utilize these carbon,such as D-trehalose,melitriose,?-D-lactose,D-galactose,sodium lactate etc.And,the napA gene of 860 and 885 bp for strain SQY5 and KSS10 were amplified,respectively.?2?Biodegradation characteristics of tetracycline/oxytetracycline were studied under various environmental conditions by using batch experiments;including inoculation dose?v/v?,initial tetracycline/oxytetracycline concentration,temperature,and pH.Response surface methodology?RSM?analysis demonstrated that the maximum degradation ratio of tetracycline and oxytetracycline could be obtained under the aerobic conditions:an initial tetracycline and oxytetracycline concentration of 61.27 and 36.31 mg?L-1,temperature of34.96 and 36.31?,pH of 7.17 and 5.45,and inoculation dose of 29.89%and 16.07%?v/v?.The results of kinetic model fitting showed that the biodegradation of tetracycline and oxytetracycline at different initial concentrations of tetracycline/oxytetracycline(10-100mg·L-1),temperatures?20-40??,and pH?5-10?accorded with the first-order degradation kinetics.When the initial concentration of tetracycline,temperature and pH were 50 mg·L-1,35?,and 8,k value were the highest,which was 0.283,0.209,and 0.176 h-1,respectively.When the initial concentration of oxytetracycline,temperature,and pH were 100 mg·L-1,35?,and 5,k value was the highest,which was 0.264,0.236,and 0.215 h-1,respectively.?3?Characteristics of aerobic biodegradation,simultaneous denitrification and nitrogen conversion of tetracycline and oxytetracycline were investigated by batch experiments.The results showed that a tetracycline reduction rate of 0.175 mg·L-1·h-1,and a maximum nitrification and denitrification rates of 1.06 and 0.83 mg·L-1·h-11 were obtained by strain SQY5,respectively.A lot of humic acid will be produced in this process,but it could be used as a carbon source in the process of nitrogen conversion.Meanwhile,C-O and C=O bond breakage may occur during tetracycline biodegradation.The removal efficiency of oxytetracycline and NH4+-N was 78.78%and 56.14%by strain KSS10 from 0 to120 h,respectively.NO3--N was almost completely converted,and the degrading process of oxytetracycline may occur demethylation and breakage of C-O and C=O bonds.The effects of tetracycline and natural algae powder as co-carbon sources on tetracycline biodegradation and nitrogen conversion were 0.11 and 1.34 mg?L-1?h-1,respectively.?4?Nine tetracycline-degradation products by strain SQY5 were identified by UPLC-Q-TOF MS?Micro?.The results showed that tetracycline biodegradation was mainly carried out by oxidation,hydrolytic ring-opening,decarbonylation,deamination,dehydroxylation,and demethylation.Nine oxytetracycline-degradation products by strain KSS10 were also identified,and the dehydration,dehydration,hydrolytic ring-opening,dehydration and reduction reaction were occurred in oxytetracycline biodegradation.Effects of tetracycline biodegradable products on the cytotoxicity of Microcystis aeruginosa were studied under the natural conditions.It showed that a high dose of products could destroy the non-enzymatic defense system of algae cells and ultimately make them completely lose the total antioxidant capacity.Bacterial SQY5 genome was also sequenced by PacBio RS II.It showed that 5006 genes was obtained.The total length and average length of genes were4,745,472 and 948 bp,respectively.The 13 gene islands were identified in bacterial SQY5contains,including hypothetical proteins,transmembrane proteins,secretory proteins,integrases,transposases and other enzymatic proteins,as well as genes related to the surface structure of cell membranes?nucleic acid binding transcription factor activity,detoxification and catalytic activity?.Besides,bacterial SQY5 also contained a variety of enzyme genes involved in tetracycline degradation and nitrogen conversion?e.g.monooxygenase,peroxidase genes etc?.Non-target metabolomic sequencing by UHPLC-QTOF-MS showed that significant differences in the metabolic processes of tetracycline degradation were found,and three KEEG pathways were also significantly increased.Meanwhile,the reaction process of cell metabolism in the process of tetracycline degradation and nitrogen conversion by strain SQY5 was deduced as follows:anabolism,cellular processes,environmental information processing,genetic information processing,catabolism.Additionally,enrichment of KEGG pathway of ABC transporters,Aminoacyl-tRNA biosynthesis and 2-Oxocarboxylic acid metabolism during tetracycline biodegradation and nitrogen conversion were significant.?5?MBBR was used to treat simulated aquaculture wastewater.The results showed that MBBR system with biological immobilized strain SQY5 had good operational stability.The removal efficiency of pollutants by biofilm can be significantly increased by moderately increasing the concentration of carbon source.And,Gammaproteobacteria,Bacteroidia,and Clostridia,Alphaproteobacteria were the most important class-level species during the operation of the system.During the operation of MBBR in stage IV and stage V,all ammonia-N was almost removed in aquaculture wastewater pre-treatment,and a degradation rate of 1.338 and 1.714 mg?L-1?d-11 were obtained,respectively.The results of this study provide a case and basis for understanding the microbial degradation mechanism of tetracycline and oxytetracycline,and also provide technical support for biological treatment of aquaculture wastewater. |
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