A directed differentiation between the biofilm and suspension was observed in the molecular microbial ecology and gene expression of different bacteria in a biofilm nitritation-anammox process operated at varying hydraulic residence times (HRT) and nitrogen loading rates (NLR). The highest degree of enrichment observed in the biofilm was of anaerobic ammonia-oxidizing bacteria (AMX) followed by that of Nitrospira spp. related nitrite-oxidizing bacteria (NOB). For AMX, a major shift from Candidatus "Brocadia fulgida" to Candidatus "Kuenenia stuttgartiensis" in both suspension and biofilm was observed with progressively shorter HRT, using discriminatory biomarkers targeting the hydrazine synthase (hzsA) gene. In parallel, expression of the hydrazine oxidoreductase gene (hzo), a functional biomarker for AMX energy metabolism, became progressively prominent in the biofilm. A marginal but statistically significant enrichment in the biofilm was observed for Nitrosomonas europaea related ammonia-oxidizing bacteria (AOB). In direct contrast to AMX, the gene expression of ammonia monooxygenase subunit A (amoA), a functional biomarker for AOB energy metabolism, progressively increased in suspension. Using gene expression and biomass concentration measures in conjunction, it was determined that signatures of AOB metabolism were primarily present in the biofilm throughout the study. On the other hand, AMX metabolism gradually shifted from being uniformly distributed in both the biofilm and suspension to primarily the biofilm at shorter HRTs and higher NLRs. These results therefore highlight the complexity and key differences in the microbial ecology, gene expression and activity between the biofilm and suspension of a nitritation-anammox process and the biokinetic and metabolic drivers for such niche segregation.

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Completely Autotrophic Nitrogen-removal Over Nitrite (CANON) moving bed biofilm reactor (MBBR) is a biofilm-based nitrogen removal system where both nitritation (ammonia oxidation to nitrite) and anaerobic ammonium oxidation (anammox) reactions occur in a single reactor under oxygen-controlled conditions. In CANON MBBR systems, the syntrophy of aerobic and anaerobic ammoniumoxidizing bacteria (AOB and AMX, respectively) is necessary to achieve stable process operation. Herein, we evaluated the spatial and temporal population and activity dynamics and niche segregation of AOB and AMX in the suspension and biofilm phases of a CANON-MBBR system. Our results demonstrated a major shift of AMX from Candidatus "Brocadia" to Candidatus "Kuenenia" during progressive reactor operation enrichment possibly due to differences in substrate affinity among these AMX guilds. In parallel, a specific differentiation in microbial N-conversion was also measured via relative expression of functional genes. Essentially, AOB activities were dominant in suspension, as inferred from the expression of ammonia monooxygenase subunit A (amoA) and AMX activities were dominant in biofilm, as inferred from the expression of hydrazine oxidase (hzo). Based on these results, it is suggested that the configuration and operating conditions in CANON MBBR systems support a systematic niche segregation of AOB and AMX ecology and activity within the biofilm and suspension phases therein.

The Completely Autotrophic Nitrogen-removal Over Nitrite (CANON) process can remove ammonia by combining partial nitritation and anaerobic ammonia oxidation (anammox) in one single reactor. Considering that the stability of CANON systems relies on the interactions between three groups of predominantly autotrophic bacteria, the effect of inorganic carbon (IC) limitation on the performance of the CANON process in a moving-bed biofilm reactor was investigated. Upon subjecting the CANON system to IC limitation, the nitrogen removal rate decreased from 80% to 44% after 27 HRTs of 50% stoichiometric IC limitation, but no significant increases in NO and N 2 O emissions were observed during the limitation. After 4 HRTs of recovery, the removal rate increased back to 62% and the gas emissions experienced a further decline. Based on these results, transient or continuous IC limitation could be deleterious to the performance of autotrophic N-removal systems and needs to be minimized to ensure stable reactor performance.