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Applied and Environmental Microbiology, August 2007, p. 4931-4939, Vol. 73, No. 15
0099-2240/07/$08.00+0     doi:10.1128/AEM.00156-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

Department of Social and Environmental Engineering, Graduate School of Engineering, Hiroshima University, 1-4-1 Kagamiyama, Higashihiroshima 739-8527, Japan,¹ Department of Urban and Environmental Engineering, Graduate School of Engineering, Hokkaido University, Sapporo 060-8628, Japan²

Received 21 January 2007/ Accepted 21 May 2007

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that "Brocadia"-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 µm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH₄⁺ and NO₂^– consumption rates decreased from 0.68 and 0.64 µmol cm^–2 h^–1 at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 µmol cm^–2 h^–1 at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH₄⁺ and NO₂^– and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.

Published ahead of print on 25 May 2007.