Discovery and metagenomic analysis of an anammox bacterial enrichment related to Candidatus “Brocadia caroliniensis” in a full-scale glycerol-fed nitritation-denitritation separate centrate treatment process

Abstract

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.

Species level enrichment of AnAOB and associated growth morphology under the effect of key metabolites

“…B. caroliniensis utilize a nirK homologue or a novel nitrite reductase employing the conventional NO-dependent pathway for hydrazine production or possess the ability to switch to a NO-dependent pathway in the absence of nitrite. An alternate pathway for NO production through oxidation of hydroxylamine by a hydroxylamine oxidoreductase ( hao )-like protein was indeed detected by Park et al (14) and proposed that this alternate pathway could potentially be activated under nitrite limitation. NO was also shown to oxidize ammonium to dinitrogen gas under nitrite limitation in Ca .…”

“…Metagenomic analysis revealed that Ca . B. caroliniensis have multiple copies of nitrite/formate transporters ( focA ) that provide a competitive advantage at low nitrite concentrations due to a low intrinsic nitrite affinity constant (14) . This could potentially help them scavenge nitrite from heterotrophic denitrifiers in the presence of organic carbon.…”

“…B. caroliniensis. (14, 35) . The significant increase in NO-dependent ammonium oxidation following nitrite limitation was concomitant with the recovery of Ca .…”

“…Attached growth of Ca . B. caroliniensis was also observed in a full-scale process treating anaerobic digester liquid supplied with glycerol as the external carbon source (14) . In this instance, the use of carriers can be used to provide a large surface area to achieve high biomass retention (40) .…”

“…Jettenia moscovienalis (2) , Ca . B. caroliniensis (14) , and Ca . B. sinica (13) were detected in distinct sidestream reactors treating anaerobic digester liquor, whereas Ca .…”