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

Anaerobic ammonium oxidation with nitrite to N-2 (anammox) is a recently di scovered microbial reaction with interesting potential for nitrogen removal from wastewater. We enriched an anammox culture from a rotating disk conta ctor (near Kolliken, Switzerland) that was used to treat ammonium-rich leac hate with low organic carbon content. This enrichment led to a relative pop ulation size of 88% anammox bacteria. The microorganism carrying out the an ammox reaction was identified by analysis of the 16S rDNA sequence and by f luorescence in situ hybridization (FISH) with 16S-rRNA-targeting probes. Th e percentage sequence identity between the 16S rDNA sequences of the Kollik en anammox organism and the archetype anammox strain Candidatus Brocadia an ammoxidans was 90.9%, but between 98.5 and 98.9% with Candidatus Kuenenia s tuttgartiensis, an organism identified in biofilms by molecular methods. Th e Kolliken culture catalyzed the anaerobic oxidation of ammonium with nitri te In a manner seemingly identical to that of Candidatus B. anammoxidans, b ut 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 obs erved only between pH 6.5 and 9, with an optimum at pH 8 and a temperature optimum at 37 degreesC. Hydroxylamine and hydrazine, which are intermediate s of the anammox reaction of Candidatus B. anammoxidans, were utilized by t he Kolliken 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-sha ped region containing ribosomes and DNA. This doughnut-shape region was obs erved with FISH as having a higher fluorescence intensity. Similar to Candi datus B. anammoxidans, the Kolliken anammox organism typically formed homog enous clusters containing up to several hundred cells within an extracellul ar matrix.