Molecular Response Characteristics Of Denitrifying Bacteria Under Oxytetracycline Stress In An Indoor Manure Soil Model

Oxytetracycline,one of the commonly used broad-spectrum tetracycline antibiotics that is mainly used for the prevention and treatment of livestock diseases.However,most of the antibiotics are released into the environment through the excretion of urine and feces in the form of prototypes or metabolites,which brings potential impact and harm to the environmental ecosystem.The long-term application of feces containing antibiotic residues to the soil may have a different effect on denitrification.However,there are still few relevant reports in this area at home and abroad.Therefore,it is necessary to evaluate the effects of oxytetracycline residue on microbial communities,especially denitrifying bacteria,in the soil environment of vegetable fields.In this study,an indoor-manure-soil model was constructed under the oxytetracycline stress.Setting up blank group(0 ?g/kg),low concentration group(20 ?g/kg),medium concentration group(200 ?g/kg)and high concentration group(2000 ?g/kg).Collecting soil samples at 1,7,14,30,and 60 days,respectively.The effects of different concentrations of oxytetracycline on soil microorganisms,especially denitrifying bacteria,were investigated using fluorescence quantitative detection,terminal restriction fragment length polymorphism analysis and high-throughput sequencing methods.Exploring the changes in abundance and diversity of major denitrifying functional genes in soil,and changes in soil microbial community structure and functional diversity.The results showed that low and medium concentrations of oxytetracycline promoted the denitrifying bacterial populations carrying the nirS and nirK genes,whereas high concentrations showed an inhibitory effect.As time increased,the abundances of nirS,nirK,and narG genes all decreased slowly.However,the strain carrying the nosZ gene responded slowly to oxytetracycline stress.Two typical denitrifying functional genes,nirS and nosZ,were analyzed by terminal restriction fragment length polymorphism.It was found that oxytetracycline had no significant effect on the dominant fragments of nirS and nosZ genes in soil.But as time went on,the denitrification fragment carrying the nirS gene changed from the 30 th to the 60 th day.This shows that oxytetracycline with different concentrations can affect the abundance of denitrifying bacteria,but it can not affect the dominant bacteria group,and other factors in the soil may have a greater impact on the dominant bacteria group.High-throughput sequencing was performed on the soil samples under OTC stress at the 1st and 30 th days.The results of microbial annotation showed that the dominant bacteria in the soil were Proteobacteria,Actinobacteria,and Firmicutes and Bacteroidetes.Proteobacteria accounted for an absolute advantage and the relative abundance decreased at the 30 th day,while the Actinobacteria had the opposite effect.It is worth noting that the Firmicutes abruptly declined on the 30 th day and turned to Bacteroidetes as the dominant phylum.At the genus level,the dominant genus on the first day was Acinetobacter,Bacillus,and Pseudomonas,and on the 30 th day,the dominant genus became Xanthomonas,Streptomyces,Stenotrophomonas.The functional annotation results showed that the carbohydrate metabolism ability of the first day was stronger than that of the 30 th day,while the concentration of oxytetracycline had no significant effect.In addition,the denitrification metabolic function was stronger on the 30 th day than on the 1st day.The relative abundance of nitrate reductase in the denitrification pathway was highest,and the relative abundance of nitrate reductase in each group of oxytetracycline addition was also higher than that in the blank group,indicating that oxytetracycline could stimulate the expression of nitrate reductase.In addition,the relative abundance of nitrite reductase and nitrous oxide reductase increased on the 30 th day,indicating that the expression of denitrification enzyme may be influenced by many factors at the same time.These results show that the long-term application of livestock manure containing low and medium concentrations of oxytetracycline will cause loss of nitrogen in the soil,which is not conducive to the growth of crops.However,high concentrations of oxytetracycline will inhibit the growth of soil microflora directly and cause damage to soil microorganisms.