The effect of substrate C/N ratio on the spatial distributions of ammonia-oxidizing bacteria and their activity was investigated by using microelectrodes with high spatial resolution and fluorescent in situ hybridization (FISH) technique. In this study, an interspecies competition for O2 between ammonia-oxidizing bacteria and heterotrophic bacteria was experimentally evaluated. An autotrophic nitrifying biofilm originally cultured at C/N=0 was used as a model biofilm to study changes in specific NH4+ oxidation rate profiles in the biofilm when the substrate C/N ratio was varied. As C/N ratio increased, specific NH4+ oxidation rates decreased in the outer part of the biofilm due to interspecies competition, while they were unchanged in the inner part. The increase in substrate C/N ratio (i.e., addition of acetate) immediately induced the interspecies competition for O2 between ammonia-oxidizing bacteria and heterotrophic bacteria at the outer part of the biofilm. As a result of the interspecies competition, NH4plus; oxidation was restrained, resulting in a decrease in the ammonia-oxidizing bacterial populations. This experimental result clearly explains the stratified spatial distributions of ammonia-oxidizing bacteria within the biofilms at higher substrate C/N ratios. The combined application of microelectrodes and FISH techniques provides new insights into microbial ecology and population dynamics of nitrifying bacteria within multi-species biofilms.

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