Comprehensive metagenomic and enzyme activity analysis reveals the inhibitory effects and potential toxic mechanism of tetracycline on denitrification in groundwater

The widespread use of tetracycline (TC) inevitably leads to its increasing emission into groundwater. However, the potential risks of TC to denitrification in groundwater remain unclear. In this study, the effects of TC on denitrification in groundwater were systematically investigated at both the protein and gene levels from the electron behavior aspect for the first time. The results showed that increasing TC from 0 to 10 µg·L−1 decreased the nitrate removal rate from 0.41 to 0.26 mg·L−1·h−1 while enhancing the residual nitrite concentration from 0.52 mg·L−1 to 50.60 mg·L−1 at the end of the experiment. From a macroscopic view, 10 µg·L−1 TC significantly inhibited microbial growth and altered microbial community structure and function in groundwater, which induced the degeneration of denitrification. From the electron behavior aspect (the electron production, electron transport and electron consumption processes), 10 µg·L−1 TC decreased the concentration of electron donors (nicotinamide adenine dinucleotide, NADH), electron transport system activity, and denitrifying enzyme activities at the protein level. At the gene level, 10 µg·L−1 TC restricted the replication of genes related to carbon metabolism, the electron transport system and denitrification. Moreover, discrepant inhibitory effects of TC on individual denitrification steps, which led to the accumulation of nitrite, were observed in this study. These results provide the information that is necessary for evaluating the potential environmental risk of antibiotics on groundwater denitrification and bring more attention to their effects on geochemical nitrogen cycles.