The simultaneous recruitment of anammox granules and biofilm by a sequential immobilization and granulation approach

Anaerobic ammonium oxidation (anammox) is an important pathway in the nitrogen cycle and for autotrophic nitrogen removal of wastewater. However, the slow granulation or biofilm formation of anammox bacteria in a continuous-flow reactor without anammox inoculum is still a challenge. To accelerate granulation or biofilm formation, an approach of sequential immobilization and granulation of anammox (SIGA) and an internal circulation immobilized blanket (ICIB) with non-granular activated sludge inoculum was investigated. Pre-immobilizing the biofilm with carriers showed higher efficiencies of nitrite removal and enrichment of Candidatus Kuenenia than anaerobic sludge without immobilization. An ICIB with pre-immobilized anaerobic biofilm and an up-flow anaerobic sludge bed (UASB) with pre-acclimated anaerobic sludge were operated for 180 d. The ICIB reactor achieved a nitrogen removal rate of 1.11 kg.N/(m(3).d) whereas the UASB reactor only obtained 0.29 kg.N/(m(3).d). Illumina HiSeq sequencing of 16S rRNA gene amplicons indicated that the relative abundance of anammox bacteria (Candidatus Kuenenia, Candidatus Brocadia, and Candidatus Jettenia) achieved 42.5-50.6% in biofilm and 45.3-47.1% in granules in the ICIB reactor. By contrast, no anammox bacteria were enriched in the sludge of the UASB reactor. These findings showed that the granulation and the biofilm formation of abundant anammox bacteria occurred in a single ICIB. The anammox biofilm and internal circulation facilitate the granulation of anammox bacteria, as the planktonic anammox cells escaped from the biofilm on the carrier provides parent cells for the formation of anammox granules under suitable hydraulic shear conditions. This study provides a new strategy for a faster start-up of anammox using activated sludge inoculum based on ICIB and SIGA.