Molecular analysis of enrichment cultures of marine ammonia oxidisers

Marine ammonia oxidising bacteria were enriched by incubation of sea water, amended with ammonium sulphate, and subsequent subculture in liquid inorganic medium. PCR primers were designed to be specific for rDNA sequences from ammonia oxidisers belonging to the β-rsub-group of the proteobacteria. These primers were then used to amplify rRNA genes from ammonia oxidiser enrichment cultures containing heterotrophs. PCR products were recovered from all cultures in which complete ammonia oxidation occurred. Subsequent rDNA sequence analysis indicated the presence of three new lineages within the clade defined by sequences of cultured β-sub-group ammonia oxidisers. Two of the new lineages showed moderate similarity to sequences from pure cultures of ammonia oxidisers previously isolated from marine and brackish environments. The third lineage (AEM-3) was deep branching and occupied an intermediate position between clades defined by Nitrosomonas or Nitrosospira, which were isolated from soil or sewage. The phylogenetic analysis suggests that, in enrichment cultures, the primers are specific for members of the target group, the β-proteobacteria ammonia oxidisers. The results also indicate the presence of previously unknown ammonia oxidisers in marine samples. The approach enabled analysis of ammonia oxidiser enrichments at an early stage and without the requirement for isolation of pure cultures, significantly reducing the time required and facilitating quantitative assessment of relatedness of strains.