Metagenomic Insights Into The Feature Of Denitrifying Molecular Response In Sediment Under Oxytetracycline Pressure

As a broad-spectrum antibiotic,oxytetracycline has a good antibacterial and growthpromoting effect and has been widely used as a feed additive in livestock and poultry breeding industry.After ingestion of oxytetracycline,only a small amount of oxytetracycline remains in animal tissues,while the majority of the excretions in the form of prototypes or metabolites enter the environment through feces and urine excretion,inevitably affecting the environment in the microbial community affect.Denitrification as an essential and essential part of nitrogen circulation in nature,plays a crucial role in alleviating eutrophication caused by excess nitrogen in water environment.However,this process is under the influence of environmental factors in the residual antibiotics and other factors.In order to evaluate the effect of oxytetracycline residues on denitrifying microbial community in water environment,the model of oxytetracycline exposure to environmental sediment in indoor water bodies was established to study the effects of oxytetracycline on the microbial community structure,And the genetic diversity,denitrification process and its major functional gene response characteristics of a systematic study.Oxytetracycline treatment group(1.0μg/L)and blank control group were set up,the sediment samples were collected at 1d、4d、16d and 30 d after administration,and the main denitrification function genes were detected by real-time quantitative PCR.The abundance of denitrification genes was analyzed by T-RFLP.The HiSeq4000 PE150 platform was used to carry out metagenomic sequencing to study the changes of flora structure and functional diversity.The results showed that exposure to oxytetracycline significantly reduced the relative abundance of nirK and nosZ genes in sediment(p <0.001),and the highest copy number of base in the blank control group was 4 folds.The addition of oxytetracycline had no significant effect on the relative abundance of nirS and narG genes.The restriction endonuclease analysis of nirS,nirK and nosZ genes showed that the addition of oxytetracycline significantly changed the community structure of nirK-type denitrifying microorganisms.In the middle and early stages of the experiment,the composition of community structure of nosZ-type denitrifying microorganisms in oxytetracycline-treated group and control group was significantly different,but in the late experimental period oxytetracycline-treated group gradually recovered to the control group.The metagenomic sequencing of the sediment samples in this experiment,the results of flora annotation showed that the dominant bacteria in the sediment were Proteobacteria,Firmicutes and Actinobacteria.The relative abundance of Proteobacteria and Actinobacteria in the oxytetracycline treatment group was significantly higher than that in the blank control group(p <0.05),whereas the relative abundance of Firmicutes was significantly lower than the blank control group(p <0.05).At the genus level,dominant bacteria in the oxytetracycline treatment group were Clostridium,Shigella,and Synechococcus.Dominant genus in the control group were Clostridium,Oscillatoria and Bayesian.Functional annotation results showed that exposure to oxytetracycline significantly reduced the abundance of functional genes involved in protein metabolism in sediments and the relative abundance of functional genes involved in denitrification in the oxytetracycline treatment group was significantly lower than that of the blank control group.At the same time,the highest abundance of nitrate reductase was found in the denitrification pathway,and the exposure to oxytetracycline significantly reduced the abundance of nitrate reductase,nitrite reductase,nitrous oxide reductase and nitrogenase but increased abundance of nitric oxide reductase.A total of 23 bacterial species were involved in the denitrification process in the functional host analysis.Pseudomonas was the only bacterial host carrying all four denitrification genes.The results of correlation analysis indicated that there was a significant correlation between the abundance of nirS gene and the abundance of Thioalkalivibrio(R = 0.733,P = 0.038).The abundance of nirK gene correlated with the abundance of Bradyrhizobium(R =-0.905,P = 0.002)and Hahella(R =-0.823,P = 0.012),while the abundance of nosZ gene was significantly and positively correlated with the abundance of Acidovorax(R = 0.785,P = 0.021),but not with Marinobacter(R =-0.753,P = 0.031)were significantly negatively correlated.These results show that oxytetracycline residues in the water environment can affect the process of denitrification in sediments,which may exacerbate the pollution of reactive nitrogen in water.