A novel start-up strategy for MBBR systems was implemented that achieved removal rates of ammonia and total nitrogen of more than 90% and 70%, respectively, with influent C/N ratio of nine. The strategy consists of operating the MBBR with non-nitrified secondary effluent (C/N=2) until ammonia removal is sustained and gradually increase the C/N ratio of the feed to the target value of nine, when the MBBR system is fed primary effluent. Before using the start-up sequence, the MBBR system treating primary effluent (C/N=9) showed negligible ammonia removal after 120 days of operation, indicating the absence of nitrifying bacteria. It was assumed that heterotrophs were outcompeting autotrophs, and the C/N ratio was decreased to allow autotrophs to proliferate. Genomic analysis indicated the presence of bacterial and fungal populations, which can perform nitrification and denitrification. The most abundant heterotrophs observed were Proteobacteria at 31%, Bacteroidetes at 22%, and Actinobacteria at 8%. The most abundant fungal population belonged to Ascomycota (10%), Basidiomycota (10%), and Mucoromycota (1%). Heterotrophic nitrification and aerobic denitrification were found to play an important role in nitrogen removal at C/N=9. Autotrophs may have proliferated at lower C/N ratio as total nitrogen removal was low, but at higher C/N a shift in microbial population should have taken place as indicated by the dominance of heterotrophs.