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Metabolic pathway of anaerobic ammonium oxidation on the basis of 15N studies in a fluidized bed reactor
Summary: A novel metabolic pathway for anaerobic ammonium oxidation with nitrite as the electron acceptor has been elucidated using 15N-Iabelled nitrogen compounds. These experiments showed that ammonium was biologically oxidized with hydroxylamine as the most probable electron acceptor. The hydroxylamine itself is most likely derived from nitrite. Batch experiments in which ammonium was oxidized with hydroxylamine transiently accumulated hydrazine. The conversion of hydrazine to dinitrogen gas is postulated as the reaction generating electron equivalents for the reduction of nitrite to hydroxylamine. During the conversion of ammonium a small amount of nitrate was formed from some of the nitrite. The addition of NH2OH to an operating fluidized bed system caused a stoichiometric increase in the ammonium conversion rate (1 mmol I−1 h−1) and a decrease in the nitrate production rate (0.5 mmol I−1 h−1). Addition of hydrazine also caused a decrease in nitrate production. On the basis of these findings it is postulated that the oxidation of nitrite to nitrate could provide the anaerobic ammonium-oxidizing bacteria with the reducing equivalents necessary for CO2 fixation.
Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor
An autotrophic synthetic medium for the enrichment of anaerobic ammonium-oxidizing (Anammox) micro-organisms was developed. This medium contained ammonium and nitrite as the only electron donor and electron acceptor respectively while carbonate was the only carbon source provided. Preliminary studies showed that the presence of nitrite and the absence of organic electron donors were essential for Anammox activity. The conversion rate of the enrichment culture in a fluidized bed reactor was 3 kg NH4 + m−3 d−1 when fed with 30 mM NH4 +. This is equivalent to a specific anaerobic ammonium oxidation rate of 1000–1100 nmol NH4 +h−1 (mg volatile solids)−1. The maximum specific oxidation rate obtained was 1500 nmol NH4 +h−1 (mg volatile solids)−1. Per mol NH4 + oxidized 0.041mol CO2 were incorporated resulting in a estimated growth rate of 0.001 h−1. The main product of the Anammox reaction is N2 but about 10% of the N-feed is converted to NO3 −. The overall nitrogen balance gave a ratio of NH4 −-conversion to NO2 −-conversion and NO3 −-production of 1:1·31±0·06:2·02±0·02. During the conversion of NH4 + with NO2 − no other intermediates or end-products such as hydroxylamine NO and N2O could be detected. Acetylene phosphate and oxygen were shown to be strong inhibitors of the Anammox activity. The dominant type of micro-organism in the enrichment culture was an irregularly shaped cell with an unusual morphology. During the enrichment for Anammox micro-organisms on synthetic medium an increase in ether lipids was observed. The colour of the biomass changed from brownish to red which was accompanied by an increase in the cytochrome content. Cytochrome spectra showed a peak at 470 nm gradually increasing in intensity during enrichment.