Toxic effect of copper ions on anammox in IFFAS process filled with ZVI-10 modified carriers

The inhibitory effects of heavy metals on anammox bacteria (AnAOB) have attracted attention worldwide. However, most are conducted in activated sludge rather than biofilm systems. The toxic effect and resistance response of anammox biofilm are not predictable from those of free-living AnAOB. Zero valent iron (ZVI) has been demonstrated to enhance anammox performance, but whether ZVI can promote AnAOB resistance to heavy metal stress remains unclear. Herein, the toxic effect of copper ions (Cu(II)) on anammox in integrated floating-film activated sludge (IFFAS) process filled with 10 wt% ZVI modified carriers (R1) was investigated. Results indicated half inhibiting concentration (IC₅₀) of Cu(II) in R1 was 9.13 mg/L, which was much higher than that in R0 filled with conventional carriers made of high density polyethylene (HDPE) (3.94 mg/L). Long-term effect of Cu(II) demonstrated that Cu(II) concentrations less than 1.0 mg/L could not inhibit anammox biofilm significantly, whereas R1 performed better anammox process than R0 under the stress of 0.1-1.0 mg/L Cu(II). The ZVI modified carriers induced more extracellular polymeric substances (EPS) to trap Cu(II) to attenuate the toxicity to AnAOB. Besides, the activities of functional enzymes related to anammox (NIR and HDH), as well as heme-c contents, were always higher in R1 than R0 regardless of the Cu(II) dosage. Candidatus Kuenenia was identified as the predominant AnAOB, which had stronger resistance to Cu(II) stress compared to other genera in the IFFAS process. Metal resistance genes (MRGs) analysis identified AnAOB induced multi-responses to resist Cu(II) stress, such as the up-regulation of copC, cutA, cutC, cutF, cueR and cueO, to synthesize more proteins with functions of copper exocytosis, conjugation and oxidation.