Aim: To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors). Methods and Results: Three bench-scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5-day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2.5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia-oxidizing bacteria, AOB; nitrite-oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB-NOB activity were less apparent, resulting in incomplete nitrification. Conclusions: Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification. Significance and Impact of the Study: This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.