Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate

Abstract.

Anaerobic ammonium oxidation with nitrite to N₂ (anammox) is a recently discovered microbial reaction with interesting potential for nitrogen removal from wastewater. We enriched an anammox culture from a rotating disk contactor (near Kölliken, Switzerland) that was used to treat ammonium-rich leachate with low organic carbon content. This enrichment led to a relative population size of 88% anammox bacteria. The microorganism carrying out the anammox reaction was identified by analysis of the 16S rDNA sequence and by fluorescence in situ hybridization (FISH) with 16S-rRNA-targeting probes. The percentage sequence identity between the 16S rDNA sequences of the Kölliken anammox organism and the archetype anammox strain Candidatus Brocadia anammoxidans was 90.9%, but between 98.5 and 98.9% with Candidatus Kuenenia stuttgartiensis, an organism identified in biofilms by molecular methods. The Kölliken culture catalyzed the anaerobic oxidation of ammonium with nitrite in a manner seemingly identical to that of Candidatus B. anammoxidans, but exhibited higher tolerance to phosphate (up to 20 mM) and to nitrite (up to 13 mM) and was active at lower cell densities. Anammox activity was observed only between pH 6.5 and 9, with an optimum at pH 8 and a temperature optimum at 37 °C. Hydroxylamine and hydrazine, which are intermediates of the anammox reaction of Candidatus B. anammoxidans, were utilized by the Kölliken organisms, and approximately 15% of the nitrite utilized during autotrophic growth was converted to nitrate. Electron microscopy showed a protein-rich region in the center of the cells surrounded by a doughnut-shaped region containing ribosomes and DNA. This doughnut-shape region was observed with FISH as having a higher fluorescence intensity. Similar to Candidatus B. anammoxidans, the Kölliken anammox organism typically formed homogenous clusters containing up to several hundred cells within an extracellular matrix.