Minimizing byproduct formation in bioelectrochemical denitrification with anammox bacteria

Autotrophic bioelectrochemical denitrification (BED) holds promise for nitrate remediation. However, the accumulation of byproducts such as NO₂^-, N₂O, and NH₄⁺, poses a significant challenge to effluent quality and climate adaptation. This study hypothesized that introducing anaerobic ammonium oxidation bacteria (anammox) to BED could alleviate this issue through synergy: a) anammox can utilize NH₄⁺ and NO₂^- from BED without producing N₂O, as seen in canonical denitrification, and b) BED can recycle NO₃^- from the anammox anabolic pathway. Results showed that Anammox_BED reduced NO₂^- accumulation by two-thirds, lowered the relative abundance of N₂O by 80 %, and eliminated NO. Metagenomic analysis revealed that the anammox species Ca. Brocadia sapporoensis tripled in abundance in the bulk sludge. Meanwhile, Pseudomonas stutzeri and Bosea robiniae, species capable of reducing nitrate via extracellular electron transfer (EET) and supplying NO₂^- to anammox, halved in relative abundance, while the abundance of Stenotrophomonas acidaminiphila, a non-EET, ammonia assimilation species, doubled following anammox introduction. Metatranscriptomic analysis found upregulation of denitrification-related functional genes in Anammox_BED biofilm and survival- and motility- related genes in bulk sludge, possibly due to insufficient substrate. Overall, BED-Anammox successfully diverted the rate-limiting EET nitrite reduction towards anammox-driven nitrite utilization thereby mitigating the generation of unwanted intermediates.