[Enrichment and identification of anaerobic ammonium oxidation bacteria]

Anaerobic ammonium oxidation (ANAMMOX) bacteria were investigated by cultivation and molecular approaches. Primary enrichments were made with NH4HCO3 and NaNO2 as substrates and two types of sludge from ANAMMOX reactors as inoculum. Two stable enrichment cultures were obtained after two years of successive transfers, and ammonium removal rate was 85%. The ANAMMOX bacteria in the enrichment cultures were identified by Planctomycetales-specific 16S rRNA gene-cloning analysis and fluorescence in situ hybridization (FISH). The clone analysis revealed that all the clonal sequences obtained from the two enrichment cultures were similar and closely related to Candidatus "Kuenenia stuttgartiensis" (99% similarities in 16S rRNA gene sequence). FISH with Planctomycetales-specific probe Pla46 and K. stuttgartiensis-specific probe Kst1275 revealed that the predominant ANAMMOX bacteria in the two enrichment cultures showed positive signals with the Pla46 and Kst1275 probes, respectively. To detect the variation in abundance of K. stuttgartiensis in the original inoculum reactor during two years of operation, FISH with Pla46 and Kst1275 probes was applied to the same sludge. The results showed that the abundance of K. stuttgartiensis in the sludge was increased from 11% to 24% within two years.