Single and combined impacts of antibiotics on aerobic denitrification: Cell growth characteristics and functional genes expressions

Antibiotic contamination has garnered significant attention, particularly given the growing pressures from aquaculture, a key contributor to environmental antibiotic loads. Addressing both antibiotic and nitrogen pollution in such ecosystems is critical. In this study, the aerobic denitrifying bacterium Marinobacter hydrocarbonoclasticus RAD-2, previously isolated in our laboratory, was subjected to a series of concentration gradients (0, 20, 40, 60, 80, 100 mg/L) to evaluate the single and combined effects of tetracycline (TET) and chlortetracycline (CTC) on the aerobic denitrification process. Among them, the combined effects of antibiotics were set up in a full-factor experimental design (a total of 30 treatment combinations) on the basis of the single-factor experiments of TET and CTC. Results demonstrated that the inhibitory impact of both antibiotics intensified with increasing concentration, with CTC exerting a more pronounced inhibitory effect. Notably, RAD-2 was unable to proliferate at 100 mg/L of TET or 80 mg/L of CTC. High concentrations of either antibiotic significantly suppressed the expression of key denitrification functional genes, including nirX, napA, norB, and nosZ. Furthermore, simultaneous exposure to both antibiotics led to a rapid decline in nitrogen removal efficiency (TET or CTC > 60 mg/L), alongside substantial inhibition of bacterial growth and functional gene expression, except for napA. Under specific concentration ranges, the combination of TET and CTC exhibits a certain degree of antagonistic effect. These findings provide critical insights into the restoration of wetland ecosystem health and inform strategies to mitigate the dual challenges of antibiotic and nitrogen pollution in aquaculture effluents.