Enrichment of anammox bacteria from mudflat sediments collected in Tokyo Bay

Abstract

We established an enrichment culture of anammox bacteria by seeding a batch reactor with sediments from the Banzu mudflat in Tokyo Bay. The reactor was intermittently fed with an anammox substrate containing NH4Cl, NaNO2 and artificial seawater (3.03% salinity) for more than 800 days. An anammox enrichment culture capable of simultaneous reduction of approximately equivalent amounts of ammonium nitrogen and nitrite nitrogen was established. Batch assays of the enrichment culture revealed that anammox activity was similar for all batches at salinities ranging from 1.01 to 3.03%, with lower activity observed at a salinity of 0.02%. We identified three 16S rRNA genes in DNA fragments amplified from the enriched culture. One of these genes, mudflat sediment clone_K01 was highly homologous with Candidatus “Kuenenia stuttgartiensis” (identity 97%) being the most abundant (85 out of isolated 94 clones). In most of the studies conducted to date, anammox species belonging to the Candidatus “Scalindua” genus have been found to be the dominant species in enrichment cultures obtained from estuarine and marine environments. The results reported here are unusual, in that, a freshwater anammox bacterium, Candidatus K. stuttgartiensis, was the dominant bacterium in a sediment sample from a marine environment. Key words: Anammox, mudflat sediment, salinity, Candidatus “Kuenenia stuttgartiensis”,Candidatus “Scalindua wagneri”