Critical role of multiple antibiotics on the denitrification rate in groundwater: Field investigative proof

The impact of antibiotics on denitrification has emerged as a significant topic; however, there is a dearth of mechanistic understanding regarding the effects of multiple antibiotics at the ng/L level on denitrification in groundwater. This study conducted five field samplings between March 2019 and July 2021 at two representative monitoring wells. The investigation utilized metagenomic sequencing to unveil the antibiotic mechanisms influencing denitrification. Results revealed the detection of 16 out of 64 antibiotics, with a maximum detection frequency and total concentration of 100 % and 187 ng/L, respectively. Additionally, both nitrate and total antibiotic concentrations exhibited a gradual decrease along the groundwater flow direction. Metagenomic evidence indicated that denitrification served as the dominant biogeochemical process controlling nitrate attenuation in groundwater. However, the denitrification capacity experienced significant inhibition in the presence of multiple antibiotics at the ng/L level. This inhibition was attributed to decreases in the relative abundance of dominant denitrifying bacteria (Candidatus_Scalindua, Herminiimonas and unclassified_p_Planctomycetes) and denitrifying functional genes (narGH, nirKS and norB), signifying the pressure exerted by antibiotics on denitrifying bacteria. The variation in antibiotic concentration (Delta Cantibiotics) indicated a change in antibiotic pressure on denitrifying bacteria. A larger Delta Cantibiotics corresponded to a greater rebound in the relative abundance of denitrifying functional genes, resulting in a faster denitrification rate (Kdenitrification). Field observations further demonstrated a positive correlation between Kdenitrification and Delta Cantibiotics. Comparatively, a higher Kdenitrification observed at higher Delta Cantibiotics was primarily due to the enrichment of more nondominant denitrifying bacteria carrying key denitrifying functional genes. In conclusion, this study underscores that multiple antibiotics at the ng/L level in groundwater inhibited denitrification, and the degree of inhibition was closely related to Delta Cantibiotics.