Rapid start-up of anaerobic ammonium oxidation (anammox) process in up-flow column reactors was successfully achieved by immobilizing minimal quantity of biomass in polyvinyl alcohol (PVA)-sodium alginate (SA) gel beads. The changes in the reactor performance (i.e., nitrogen removal rate; NRR) were monitored with time. The results demonstrate that the reactor containing the immobilized biomass concentration of 0.33 g-VSS L(-1) achieved NRR of 10.8 kg-N m(-3) d(-1) after 35-day operation, whereas the reactor containing the granular biomass of 2.5 g-VSS L(-1) could achieve only NRR of 3.5 kg-N m(-3) d(-1). This indicates that the gel immobilization method requires much lower seeding biomass for start-up of anammox reactor. To explain the better performance of the immobilized biomass, the biological and physicochemical properties of the immobilized biomass were characterized and compared with the naturally aggregated granular biomass. Effective diffusion coefficient (De) in the immobilized biomass was directly determined by microelectrodes and found to be three times higher than one in the granular biomass. High anammox activity (i.e., NH4(+) and NO2(-) consumption rates) was evenly detected throughout the gel beads by microelectrodes due to faster and deeper substrate transport. In contrast, anammox activity was localized in the outer layers of the granular biomass, indicating that the inner biomass could not contribute to the nitrogen removal. This difference was in good agreement with the spatial distribution of microbes analysed by fluorescence in situ hybridization (FISH). Based on these results, PVA-SA gel immobilization is an efficient strategy to initiate anammox reactors with minimal quantity of anammox biomass.

The identification of the key nitrous oxide (N2O) production pathways is important to establish a strategy to mitigate N2O emission. In this study, we combined real-time gas-monitoring analysis, (15)N stable isotope analysis, denitrification functional gene transcriptome analysis and microscale N2O concentration measurements to identify the main N2O producers in a partial nitrification (PN) aerobic granule reactor, which was fed with ammonium and acetate. Our results suggest that heterotrophic denitrification was the main contributor to N2O production in our PN aerobic granule reactor. The heterotrophic denitrifiers were probably related to Rhodocyclales bacteria, although different types of bacteria were active in the initial and latter stages of the PN reaction cycles, most likely in response to the presence of acetate. Hydroxylamine oxidation and nitrifier denitrification occurred, but their contribution to N2O emission was relatively small (20-30%) compared with heterotrophic denitrification. Our approach can be useful to quantitatively examine the relative contributions of the three pathways (hydroxylamine oxidation, nitrifier denitrification and heterotrophic denitrification) to N2O emission in mixed microbial populations.

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