Effect of inoculated sludge concentration on start-up of anammox reactor: Nitrogen removal performance and metabolic pathways

Abstract

The anammox process is efficient for nitrogen removal but faces challenges due to slow bacterial growth and limited inoculated sludge supply. This study examined the effects of different inoculated sludge concentrations (3.5, 7, and 14 g/L) on start-up and nitrogen metabolism in anammox reactors. Three identical reactors were operated under controlled conditions, with comprehensive analysis of nitrogen removal efficiency, sludge characteristics, and microbial community dynamics through metagenomic and transcriptomic approaches. Results demonstrated that higher inoculated sludge concentrations accelerated reactor start-up, with the 14 g/L reactor achieving stable operation in 13 days compared to 44 days for the 3.5 g/L reactor. However, the improvement in nitrogen removal rate showed a boundary effect, not proportional to the increase in sludge concentration. Notably, reactors with higher inoculated sludge concentrations exhibited lower sludge loads but higher sludge yield coefficients. Metagenomic analysis revealed Candidatus Kuenenia as the dominant anammox bacteria, with decreasing hydrazine dehydrogenase (hdh) gene expression levels observed at higher sludge concentrations, suggesting hydrazine synthesis as a potential rate-limiting step. This study provides novel insights into the optimal range of inoculated sludge concentration for anammox reactor start-up and elucidates the underlying metabolic mechanisms, offering valuable guidance for practical engineering applications.