A distinctive red biofilm was observed in a glycerol-fed digester liquid effluent treatment process coupling partial nitrification (nitritation) and partial denitrification (denitritation) processes. Based on initial phylogenetic screening using 16S rRNA clone libraries and quantitative polymerase chain reaction, the biofilm was enriched in novel anaerobic ammonium oxidizing bacteria (AMX/anammox) closely related to Candidatus “Brocadia caroliniensis”. The metabolic functionality of the C. “Brocadia caroliniensis” enrichment was further explored using high-throughput sequencing and de novo metagenome assembly. The population anammox genome that was binned from the metagenome consisted of 209 contigs with a total of 3.73 Mbp consensus sequences having 43.3% GC content, and 27.4 average coverage depth. The assembled metagenome bin was comprised of 3582 open reading frames (ORFs). Based on 16S rRNA similarity the binned metagenome was closely related with Candidatus “Brocadia caroliniensis”, Candidatus “Brocadia fulgida”, planctomycete KSU-1, and Candidatus “Kuenenia stuttgartiensis” with 99%, 96%, 92% and 93% similarity, respectively. Essential genes in anammox metabolic functions including ammonium and nitrite transport, hydrazine synthesis, electron transfer for catabolism, and inorganic carbon fixation, among several other anabolic pathways, were also observed in the population genome of the C. “Brocadia caroliniensis” related enrichment. Our results demonstrate the wider profusion of anammox bacteria in engineered nitrogen removal systems than expected. The utility of metagenomics approaches to deciphering such novel functionality in these systems is also highlighted.@en