Metagenomics insight into the long-term effect of ferrous ions on the mainstream anammox system

Anaerobic ammonium oxidation (anammox) bacteria have a high requirement for iron for their growth and metabolism. However, it remains unclear whether iron supplementation can sustain the stability of mainstream anammox systems at varying temperatures. Here, we investigated the long-term effects of Fe²⁺ on the mainstream anammox systems. Our findings revealed that the nitrogen removal efficiency (NRE) of the anammox system supplemented with 5 mg/L Fe²⁺ decreased from 76.5 ± 0.76% at 35 °C to 39.0 ± 9.9% at 25 °C. Notably, higher dosages of Fe²⁺ (15 mg/L and 30 mg/L) inhibited the anammox system, resulting in NREs of 15.9 ± 8.1% and 2.5 ± 1.1% at 25 °C, respectively. The results of microbial communities and function profiles suggested that the high Fe²⁺ dosage seriously affected the iron assimilation and utilization in the mainstream anammox system. This was evident from the decreased abundance of genes associated with Fe(II) transport and uptake, which in turn hindered the biosynthesis of intracellular iron-cofactors, resulting in decrease in the absolute abundance of Candidatus Brocadia, a key anammox bacterium, as well as a decline in NRE. Furthermore, our results showed that the anammox process was more susceptible to iron supplementation at 25 °C compared to 35 °C, which may be due to the oxidative stress reactions induced by combined lowered temperature and a high Fe²⁺ dosage. Overall, these findings offer a deeper understanding of the effect of iron in mainstream anammox systems, which can contribute to improved stability maintenance and effectiveness of anammox processes.