Potential effects of Cu²⁺ stress on nitrogen removal performance, microbial characteristics, and metabolism pathways of biofilm reactor

Sequencing batch biofilm reactors (SBBR) were utilized to investigate the impact of Cu²⁺ on nitrogen (N) removal and microbial characteristics. The result indicated that the low concentration of Cu²⁺ (0.5 mg L⁻¹) facilitated the removal of ammonia nitrogen (NH₄⁺-N), total nitrogen (TN), nitrate nitrogen (NO₃⁻-N), and chemical oxygen demand (COD). In comparison to the average effluent concentration of the control group, the average effluent concentrations of NH₄⁺-N, NO₃⁻-N, COD, and TN were found to decrease by 40.53%, 17.02%, 10.73%, and 15.86%, respectively. Conversely, the high concentration of Cu²⁺ (5 mg L⁻¹) resulted in an increase of 94.27%, 55.47%, 22.22%, and 14.23% in the aforementioned parameters, compared to the control group. Low concentrations of Cu²⁺ increased the abundance of nitrifying bacteria (Rhodanobacter, unclassified-o-Sacharimonadales), denitrifying bacteria (Thermomonas, Comamonas), denitrification-associated genes (hao, nosZ, norC, nffA, nirB, nick, and nifD), and heavy-metal-resistant genes related to Cu²⁺ (pcoB, cutM, cutC, pcoA, copZ) to promote nitrification and denitrification. Conversely, high concentration Cu²⁺ hindered the interspecies relationship among denitrifying bacteria genera, nitrifying bacteria genera, and other genera, reducing denitrification and nitrification efficiency. Cu²⁺ involved in the N and tricarboxylic acid (TCA) cycles, as evidenced by changes in the abundance of key enzymes, such as (EC:1.7.99.1), (EC:1.7.2.4), and (EC:1.1.1.42), which initially increased and then decreased with varying concentrations of Cu²⁺. Conversely, the abundance of EC1.7.2.1, associated with the accumulation of nitrite nitrogen (NO₂⁻-N), gradually declined. These findings provided insights into the impact of Cu²⁺ on biological N removal.