A synthetic medium was developed to enrich anaerobic ammonium-oxidizing (Anammox) microorganisms for autotrophic growth in a fluidized bed reactor. The medium contained ammonium and nitrite as electron donors and acceptors, respectively, and carbonate as the sole carbon source. The Anammox process was found to be essential for the conversion of ammonium to dinitrogen gas, with nitrite acting as a key electron acceptor. The specific oxidation rate of the enrichment culture was 1000–1100 nmol NH₄⁺ h⁻¹ (mg volatile solids)⁻¹, with a maximum of 1500 nmol NH₄⁺ h⁻¹ (mg volatile solids)⁻¹. The process incorporated 0.041 mol CO₂ per mol NH₄⁺ oxidized, resulting in a growth rate of 0.001 h⁻¹. The main product of the Anammox reaction was N₂, but about 10% of the nitrogen feed was converted to NO₃⁻. The nitrogen balance showed a ratio of NH₄⁺-conversion to NO₂⁻-conversion and NO₃⁻-production of 1:1.31 ± 0.06:0.22 ± 0.02. The Anammox process did not produce intermediates such as hydroxylamine, NO, or N₂O. Acetylene, phosphate, and oxygen were strong inhibitors of Anammox activity. The dominant microorganism in the enrichment culture was an irregularly shaped cell with unusual morphology. The culture showed an increase in ether lipids and a shift in biomass color from brown to red, accompanied by an increase in cytochrome content. Cytochrome spectra showed a peak at 470 nm, which gradually increased in intensity during enrichment. The Anammox process was found to be autotrophic, with bicarbonate as the sole carbon source. The process was efficient in converting ammonium to dinitrogen gas, with a conversion rate of 3 kg NH₄⁺ m⁻³ d⁻¹ when fed with 30 mM NH₄⁺. The process was compared to other nitrogen removal methods, showing its potential for nitrogen removal from wastewater with low carbon content. The study also identified the presence of nitrifiers in the Anammox sludge, suggesting their ability to survive long periods of anaerobiosis. The findings indicate that the observed increase in Anammox capacity is directly related to an increase in morphologically conspicuous microorganisms, ether lipids, and cytochromes. These properties may be due to a single organism or multiple organisms responsible for the Anammox reaction. The study highlights the potential of the Anammox process for nitrogen removal in wastewater treatment.A synthetic medium was developed to enrich anaerobic ammonium-oxidizing (Anammox) microorganisms for autotrophic growth in a fluidized bed reactor. The medium contained ammonium and nitrite as electron donors and acceptors, respectively, and carbonate as the sole carbon source. The Anammox process was found to be essential for the conversion of ammonium to dinitrogen gas, with nitrite acting as a key electron acceptor. The specific oxidation rate of the enrichment culture was 1000–1100 nmol NH₄⁺ h⁻¹ (mg volatile solids)⁻¹, with a maximum of 1500 nmol NH₄⁺ h⁻¹ (mg volatile solids)⁻¹. The process incorporated 0.041 mol CO₂ per mol NH₄⁺ oxidized, resulting in a growth rate of 0.001 h⁻¹. The main product of the Anammox reaction was N₂, but about 10% of the nitrogen feed was converted to NO₃⁻. The nitrogen balance showed a ratio of NH₄⁺-conversion to NO₂⁻-conversion and NO₃⁻-production of 1:1.31 ± 0.06:0.22 ± 0.02. The Anammox process did not produce intermediates such as hydroxylamine, NO, or N₂O. Acetylene, phosphate, and oxygen were strong inhibitors of Anammox activity. The dominant microorganism in the enrichment culture was an irregularly shaped cell with unusual morphology. The culture showed an increase in ether lipids and a shift in biomass color from brown to red, accompanied by an increase in cytochrome content. Cytochrome spectra showed a peak at 470 nm, which gradually increased in intensity during enrichment. The Anammox process was found to be autotrophic, with bicarbonate as the sole carbon source. The process was efficient in converting ammonium to dinitrogen gas, with a conversion rate of 3 kg NH₄⁺ m⁻³ d⁻¹ when fed with 30 mM NH₄⁺. The process was compared to other nitrogen removal methods, showing its potential for nitrogen removal from wastewater with low carbon content. The study also identified the presence of nitrifiers in the Anammox sludge, suggesting their ability to survive long periods of anaerobiosis. The findings indicate that the observed increase in Anammox capacity is directly related to an increase in morphologically conspicuous microorganisms, ether lipids, and cytochromes. These properties may be due to a single organism or multiple organisms responsible for the Anammox reaction. The study highlights the potential of the Anammox process for nitrogen removal in wastewater treatment.