Anaerobic Ammonium Oxidation (Anammox) with Planktonic Cells in a Redox-Stable Semicontinuous Stirred-Tank Reactor

Anaerobic ammonium oxidizing (anammox) bacteria are routinely cultivated in mixed culture in biomass-retaining bioreactors or as planktonic cells in membrane bioreactors. Here, we demonstrate that anammox bacteria can also be cultivated as planktonic cells in a semicontinuous stirred-tank reactor (semi-CSTR) with a specific growth rate mu of 0.33 d(-1) at 30 degrees C. Redox potential inside the reactor stabilized at around 10 mV (+/- 15 mV; vs standard hydrogen electrode) without gas purging. Reactor headspace pressure was used as a sensitive and real-time indicator for nitrogen evolution and anammox activity. The reactor was dominated by an organism closely related to Candidatus Kuenenia stuttgartiensis (similar to 87% abundance) as shown by Illumina amplicon sequencing and fluorescence in situ hybridization. Epifluorescence microscopy demonstrated that all cells were in their planktonic form. Mass balance analysis revealed a nitrite/ammonium ratio of 1.270, a nitrate/ammonium ratio of 0.238, and a biomass yield of 1.97 g volatile suspended solids per mole of consumed ammonium. Batch experiments with the reactor effluent showed that anammox activities were sensitive to sulfide (IC50 = 5 mu M) and chloramphenicol (IC50 = 19 mg L-1), much lower than reported for granular anammox biomass. This study shows that semi-CSTR is a powerful tool to study anammox bacteria.