A first assessment of anammox activity, which is a unique N2 emission process, was conducted in samples of coastal marine sediment from Japan with a 15 N tracer. The occurrence and diversity of bacteria possibly responsible for the anammox process were also evaluated by selective PCR-amplification of the 16S rRNA gene for known anammox bacteria. Anammox activity, detected by measuring 14 N 15 N gas production, was only found in samples collected at the intertidal sand bank located at the Yodo River estuary. In the Yodo River samples, 16S rRNA gene fragments affiliated with the known anammox bacterial lineage were also recovered, and the two major phylotypes were both "Candidatus Scalindua wagneri" relatives with 95% and 98% sequence similarity. Even from the other samples in which no recognizable anammox activity was detected, 16S rRNA gene fragments related to known anammox bacteria, but not to "Ca. S. wagneri", were detected. This is the first report of anammox-mediated N2 emission in coastal marine environments in Japan. Notably, the PCR-based analysis allowed us to discover unexpected phylogenetic diversity of anammox bacteria-related 16S rRNA gene sequences. The selective PCR primer set developed in this study could be a powerful tool to unveil the ecology of anammox bacteria in natural environments.

Although the emission of N2 via anaerobic ammonium oxidation (anammox) is a key process in the elimination of nitrogenous compounds from aquatic environments, little information is available regarding its significance and the relevant microorganisms (anammox bacteria) in eutrophic freshwater lakes. In the present study, the anammox bacteria in the sediment of a eutrophic lake in Japan, Lake Kitaura, were examined using a 15 N-tracer technique to measure their potential anammox activity. Potential anammox activity was localized to the northern region of the lake where a stable supply of both NH4 + and NO3− existed in the sediment. These results suggest the contribution of anammox bacteria to the total emission of N2 from sediment in this eutrophic lake to not be negligible. Moreover, selective PCR successfully amplified anammox bacteria-related (Brocadiales-related) 16S rRNA genes from sediment samples in which potential anammox activity was observed. The clone libraries consisted of diverse phylotypes except the genus "Scalindua"-lineages, and the lineages of genus "Brocadia" were dominantly recovered, followed by the genus "Kuenenia"-lineages. Most of them, however, were novel and phylogenetically distinguishable from known Brocadiales species. A unique population of anammox bacteria inhabits and potentially contributes to the emission of N2 from Lake Kitaura.

Mangrove forests are common in subtropical regions, and have received considerable attention as vegetative buffers against anthropogenic N-loading. In this study, we investigated anaerobic ammonium oxidation (anammox) as one of potentially important microbial N-removing pathways in mangrove and shrimp pond sediment in Haiphong, Vietnam. Measurements with 15 N-labeled compounds demonstrated the occurrence of anammox in sediment of mangrove forest and a water channel connecting shrimp ponds to the sea in both 2005 and 2007, and of a semi-intensive shrimp pond in 2005. The rate of potential anammox activity reached to 0.7 nmol-N2 cm −3 h −1 , although the contribution of anammox was less significant than denitrification. Anammox-type 16S rRNA gene fragments phylogenetically related to 'Scalindua' species were predominantly recovered from mangrove forest and water channel sediment in a PCRclone library analysis targeting anammox bacteria. 'Kuenenia'-like gene fragments were also recovered from shrimp pond sediment as the major component. We demonstrated the occurrence of potential anammox activity, and suggested the possibility that diverse species of uncultured anammox bacteria contribute to the nitrogen cycle in subtropical mangrove-aquaculture ecosystems. Furthermore, this study provides new insight into the biogeography of anammox bacteria: 'Scalindua' and 'Kuenenia'-like species coexisted in the blackish sediment as in some temperate estuarine sediment.

Twenty-five replicates of growth medium for anaerobic ammonium oxidation (anammox) containing 15 N-labeled ammonium and non-labeled nitrite were inoculated into an anammox enrichment culture at low density, and anaerobically incubated batchwise. In the headspace, 29 N 2 partial pressure linearly increased via anammox in 25 vials, confirming that anammox populations were viable in all subcultures. On prolonged incubation, exponential increases in 29 N 2 were not observed in all but 13 subcultures, suggesting that the anammox population may not proliferate unless all conditions for growth are satisfied. The estimated first-order rate coefficients in those 13 subcultures varied from 0.0029 to 0.0048 h −1 .

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