Predation of nitritation–anammox biofilms used for nitrogen removal from wastewater

Predation is assumed to be a major cause of bacterial mortality in wastewater treatment plants (WWTP). Grazing on the slowly growing autotrophic ammonia oxidizing bacteria (AOB) and anaerobic ammonium oxidizing bacteria (AMX) may result in loss of biomass, which could compromise nitrogen removal by the nitritation-anammox process. However, predation, particularly of anaerobic AMX, is unknown. We investigated the presence of protozoa, AOB and AMX and the possible predation in nitritation-anammox biofilms from several WWTPs. By fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM), predator and prey were localized in intact biofilm cryosections. Different broad morphological types of protozoa were found at different biofilm depths. Large variations in abundance of protozoa were seen. One reactor showed a predation event of amoeba-like protozoa, forming grazing fronts reaching deep biofilm regions that were dominated by the anaerobic AMX. Both AOB and AMX were grazed by the amoeba, as revealed by FISH-CLSM. Hence, even AMX, living in the deeper layers of stratified biofilms, are subjected to predation. Interestingly, different colocalization was observed between the amoeba-like protozoa and two different Ca. Brocadia AMX sublineages, indicating different grazing patterns. The findings indicate that predation pressure can be an important factor regulating the abundance of AOB and AMX, with implications for nitrogen removal from wastewater.