Fluorescence in-situ hybridization (FISH) and DO microelectrodes were applied to biofilms developed in a novel reactor named DHS (downflow hanging sponge-cubes), to investigate the mechanisms of simultaneous carbon removal and nitrification. The DHS reactor was employed as an aerobic post-treatment process after an UASB anaerobic pre-treatment process receiving a municipal sewage. The presence ratio of Nitrosomonas and Nitrobacter cells to total cells of the DHS biomass was estimated by FISH technique to be 1.4% and 0.18%, respectively. Cell concentrations of both nitrifying bacteria were in good agreement with the magnitudes of ammonia-oxidizing and nitrite-oxidizing activities evaluated from batch tests. The habitats of both nitrifiers were the interior space of sponge-cubes, rather than within the biofilms attached onto sponge-cube surfaces. DO microelectrodes verify that the sponge-cubes insides were kept aerobically throughout the whole reactor height excluding the inlet vicinity portion.

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