Fate of 14C-labeled microbial products derived from nitrifying bacteria in autotrophic nitrifying biofilms

The cross-feeding of microbial products derived from C-14-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [C-14] bicarbonate, biofilm samples were incubated with and without NH4+ as a sole energy source for 10 days. The transfer of C-14 originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the beta-Proteobacteria showed significant uptake of C-14-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH4+ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized C-14-labeled products in the culture with NH4+ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of C-14-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.