Stepwise Cathodic Potential as a Double-Edged Sword for Anammox: Enzyme-Level Inhibition versus Multi-Pathway Compensation in a Biocathode

Abstract Coupling anammox with bioelectrochemical systems (BESs) offers carbon-free autotrophic nitrogen removal, yet the cathodic potential required for NO 3 ⁻ reduction to NO 2 ⁻ (+ 0.433 V vs. SHE) lies far above the activity window of anaerobic ammonium-oxidizing bacteria (AnAOB), creating a fundamental potential gap. Here, anammox biocathodes in dual-chamber BESs were operated under nine stepwise potentials (+ 0.105 to + 0.433 V vs. SHE, with a + 0.000 V control), each alternated with + 0.433 V in 30-min segments over 3-h cycles, and interrogated through short-term kinetics, 16S rRNA sequencing, and proteomics. Nitrogen removal peaked at + 0.145 V (NH 4 + -N 94.1%, NO 2 ⁻ -N 96.2%), declined to a valley at + 0.345 V, and rebounded to 46.7% at + 0.385 V as non-anammox pathways compensated for impaired anammox activity. Charge integration of the I–t curves showed that the stepwise cathodic potential acted as a regulatory signal rather than an electron source. The dominant AnAOB genus Candidatus Brocadia peaked in abundance (7.96%) at + 0.305 V even as activity was suppressed. Nitrification proteins hyperactivated at + 0.265 ± 0.04 V alongside excess NO 3 ⁻ accumulation, while upregulation of the DNRA protein NrfC implied internal ammonium recycling at high potentials. Hence, a two-stage stepwise-potential-assisted anammox denitrification (SPAD) strategy (+ 0.145 V × 90 min → +0.385 V × 30 min) was proposed to convert the potential gap into a temporal potential relay for autotrophic nitrogen removal.