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 degrees 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.

Nitrogen removal through different pathways in an aged refuse bioreactor treating mature landfill leachate

“…However, its density was very low. Candidatus Kuenenia stuttgartiensis is a salt-and alkaline-resistant bacterium Strous et al 2006), and it was detected mostly in wastewater treatment system (Egli et al 2001;Schmid et al 2000). Previous research showed that anammox diversity was usually low in an ecosystem (Schmid et al 2007;Kartal et al 2007).…”

“…qPCR assays were performed using SYBR Green I and conducted in FTC2000 fluorescence real-time PCR system (Canada). Primer pairs included Bacteria1055f (5′-ATGGCTGTCGTCAGCT-3′) and Bacteria1392r (5′-AC GGGCGGTGTGTAC-3′) for total bacterial 16S rRNA genes (Egli et al 2001), amoA-1 F (5′-GGGGTTTCTACTGGTG GT-3′) and amoA-2R (5′-CCCCTCKGSAAAGCCTTCTTC-3′) for AOB amoA genes (Pynaert et al 2003), and anammox-1 (5′-GGATTAGGCATGCAAGTC-3′) and anammox-2 (5′-TCTGTATTACCGCGGCT-3′) for anammox bacteria 16S rRNA genes (Tal et al 2006;Hu et al 2010). The 50-μL PCR reaction contained 25 μL of 2× PCR buffer, 1 μL of each primer (25 μM), 0.5 μL of SYBR Green I fluorescent dye, 2 μL of DNA template, and DEPC water.…”

Start-up of Autotrophic Nitrogen Removal Reactors via Sequential Biocatalyst Addition

“…The dominant AAOB found in the RBC biofilm (accession number AF525953) showed 98% sequence similarity with that present in a full-scale autotrophic N removing RBC reactor treating an NH4 + -rich leachate (7). To provide further evidence that the abundant planctomycetes in the RBC biofilm were responsible for the anammox reaction, an anoxic fixed film bioreactor on NH4 + /NO2 -was started with carrier material that was submerged in the RBC reactor for 1 month prior to its use in the reactor.…”

Landfill leachate treatment with a novel process: Anaerobic ammonium oxidation (Anammox) combined with soil infiltration system

“…(1)) [12]. Without the need of organics, this technology has been developed to mainly treat ammonium rich wastewaters, such as landfill leachate, poultry manure and supernatant of digested sludge [13][14][15][16]. …”