A comprehensive insight into the functional bacteria and genes and their roles in simultaneous denitrification and anammox system at varying substrate loadings

Simultaneous denitrification and anammox process are newly developed wastewater treatment processes with high-nitrogen removal efficiency, but little information is available regarding its optimal operational conditions and molecular mechanisms. In this study, a lab-scale up-flow anaerobic sludge blanket bioreactor fed with nitrate and ammonia was continuously operated for 180days to establish the simultaneous denitrification and anammox system, so as to investigate the changing patterns of nitrogen removal efficiency and functional bacteria and genes at varying substrate loadings. Results showed that the optimal removal efficiency of total nitrogen (TN) reached 92.47%, corresponding to TN loading removal rate of 0.9kg-Nm(-3)day(-1) that was achieved at low influent substrate concentrations and short hydraulic retention time (HRT) (4h). High-throughput sequencing of 16S rRNA gene amplicons revealed that the predominant denitrifying and anammox bacteria in the coupling system were Thauera (relative abundance of 25.30%) and Candidatus Brocadia (relative abundance of 6.85%), respectively. Quantitative real-time PCR showed that the anammox genes (hzsA, hzsB, and hzo) and denitrifying genes (narG, napA, and nirS) demonstrated high abundance in the optimized bioreactor. Batch experiments were also conducted to determine the temporal difference in the expression of the anammox and denitrifying genes. NarG was found to play a crucial role in nitrate reduction to produce nitrite for anammox bacteria, while nirS was identified as the key genes for nitrite reduction to produce nitric oxide, which acted as an important role in both denitrification and anammox reaction.