Granulation of a planktonic thermophilic anammox culture enables high rate nitrogen removal from warm nitrogen rich wastewater: A proof of concept

Abstract

Anaerobic ammonium oxidation (anammox) process has been widely studied for nitrogen removal from organic carbon-deficient wastewaters at mesophilic conditions (<40 °C). While two recent studies have reported the enrichment of thermophilic anammox communities, their high-rate nitrogen removal potential in scalable reactors remains unexplored, which is critical to treat warm nitrogen-rich wastewaters (45-60 °C). This study illustrates the first successful cultivation of granular thermophilic anammox sludge utilizing a planktonic enrichment culture as inoculum, demonstrating the highest thermophilic nitrogen removal rates in an expanded granular sludge bed reactor at 50 °C. A stepwise increase in hydraulic upflow velocity (HUV) from 2 to 10 m/h and nitrogen loading rate (NLR) from 0.24 to 1.11 kgN/m³/d promoted the transition from planktonic growth to coarse aggregates, while a sudden reduction in HUV to 0.2 m/h triggered their transformation into stable granules, showing high specific anammox activity (0.8–1 gNH₄⁺-N/gVSS/d) and morphological characteristics similar to their mesophilic counterparts. Granulation enabled high biomass accumulation, achieving a steady state nitrogen removal rate of 8.57 ± 0.05 kg N/m³/d, the highest reported for thermophilic anammox. Microbial community analysis revealed that Candidatus Brocadia remained the dominant anammox genus, with a high relative abundance of 64–80% during granulation. Structural extracellular polymeric substances (EPS) from thermophilic anammox granules showed higher extraction yields than mesophilic counterparts, containing β-sheet-rich proteins as the predominant fraction. These findings provide technical insights into anammox granulation utilizing planktonic cells and establish a foundation for developing thermophilic anammox-based processes for treating warm nitrogen-rich wastewaters such as, reject water from thermophilic anaerobic digestion of sewage sludge.