Dissimilatory nitrate reduction to ammonium-induced false stability masks dysfunctional Candidatus Brocadia failure during ANAMMOX start-up from municipal activated sludge.

Enriching anaerobic ammonium oxidation (ANAMMOX) bacteria from conventional municipal activated sludge remains difficult despite reported feasibility, particularly without highly enriched anammox cultures (HeAnAC). This study examined ANAMMOX start-up in an anaerobic-membrane-bioreactor fed ammonium (NH4+) and nitrite (NO2-) under organic carbon-free conditions. Initial nitrogen removal (NH4+: 78%; NO2-: 98%) declined sharply after day 70 (<11% and <28%, respectively), coinciding with stable hydrazine synthase gene (hzs) abundance, elevated DNRA nitrite reductase gene (nrfA) abundance, and higher enzyme activity (NrfA). Metagenomic and enzymatic analyses indicated that dissimilatory nitrate reduction to ammonium (DNRA) bacteria temporarily compensated for impaired Candidatus Brocadia, masking collapse. Prolonged DNRA dominance disrupted the NH4+/ NO2- ratio, destabilized biomass, and induced excess soluble microbial products (SMP) and extracellular polymeric substances (EPS). These changes ultimately led to late-stage NO2- removal decline and a shift from supportive to inhibitory effects on ANAMMOX. Monitoring nrfA and hzs may allow early detection of DNRA dominance.