Microautoradiography combined with fluorescent in situ hybridization (MAR-FISH), a powerful tool for linking physiology with identification of individual cells, was applied to investigate microbial interactions between nitrifying bacteria and coexisting heterotrophic bacteria in an autotrophic nitrifying biofilm community fed with only ammonia as the sole energy source and bicarbonate as the sole carbon source. First, nitrifying bacteria were radiolabeled by culturing the biofilm samples with [14C]bicarbonate for 6 h, and then the transfer of radioactivity from nitrifying bacteria to heterotrophic bacteria was monitored by using MAR-FISH. MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse. We could obtain direct evidence that organic matter derived from nitrifiers was subsequently utilized by mainly filamentous bacteria belonging to the Chloroflexi (green non-sulfur bacteria) group or CFB group in the biofilm, which was clearly visualized by MAR-FISH at single cell resolution for the first time. On the other hand, the members of the α- and γ-Proteobacteria were specialized to utilize low-molecular-weight organic matter. This community represents functionally integrated units that assure maximum access to and utilization of metabolites of nitrifiers.

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