Hydroxylamine is a key intermediate in several biological reactions of the global nitrogen cycle. However, the role of hydroxylamine in anammox is still not fully understood. In this work, the impact of hydroxylamine (also in combination with other substrates) on the metabolism of a planktonic enrichment culture of the anammox species Ca. Kuenenia stuttgartiensis was studied. Anammox bacteria were observed to produce ammonium both from hydroxylamine and hydrazine, and hydroxylamine was consumed simultaneously with nitrite. Hydrazine accumulation - signature for the presence of anammox bacteria - strongly depended on the available substrates, being higher with ammonium and lower with nitrite. Furthermore, the results presented here indicate that hydrazine accumulation is not the result of the inhibition of hydrazine dehydrogenase, as commonly assumed, but the product of hydroxylamine disproportionation. All kinetic parameters for the identified reactions were estimated by mathematical modelling. Moreover, the simultaneous consumption and growth on ammonium, nitrite and hydroxylamine of anammox bacteria was demonstrated, this was accompanied by a reduction in the nitrate production. Ultimately, this study advances the fundamental understanding of the metabolic versatility of anammox bacteria, and highlights the potential role played by metabolic intermediates (i.e. hydroxylamine, hydrazine) in shaping natural and engineered microbial communities.@en