Low-level cadmium alleviates the disturbance of doxycycline on nitrogen removal and N2O emissions in ditch wetlands by altering microbial community and enzymatic activity

Excessive use of feed additives and chemical fertilizers leads to the accumulation of antibiotics and heavy metals in water environments. Studies have reported the toxicity of combined pollutants on the performance and mi-crobial community in sequencing batch reactors. However, the influence of combined pollutants on nitrogen removal in wetlands remains unclear. Here, an 84 d exposure experiment was conducted to investigate the effects of cadmium (Cd) and doxycycline (DC) on nitrogen metabolism in ecological ditches using metagenomic anal-ysis. The results showed that adding 50 mg L-1 DC at 5 mg L-1 Cd stress deteriorated nitrogen removal per-formance and significantly increased N2O emissions and lactate dehydrogenase release. However, a low level of Cd (0.5 mg L-1) significantly reduced N2O emissions under the high DC concentration pressure (50 mg L-1). The increasing Cd concentration markedly weakened the inhibition of DC (1 mg L-1) on the relative abundances of the dominant genera. Moreover, high concentrations of mixed Cd/DC showed stronger inhibitory effects on glucokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. NADH had a higher tolerance to mixed Cd/DC than ATP. However, low levels of mixed Cd/DC led to a higher abundance of key denitrifying enzymes and genes. The assimilatory nitrate reduction pathway increased under the mixture of Cd/DC stress, accompanied by an increase in ferredoxin-nitrite/nitrate reductase and the abundance of nasA and nirA genes. Our results indicate that the presence of low-level Cd could alleviate the impact of DC on nitrogen removal and N2O emissions in ecological ditches by altering microbial community and enzymatic activity associated with transport, metabolism, and gene expression. However, the increasing Cd concentration at DC stress could intensify the combined toxicity. Overall, our findings provide new insights into the ecological toxicity of emerging combined pollutants and their impact on biological nitrogen transformation in wastewater treatment systems.