Abstract:Anammox autotrophic denitrification process has significantly low-carbon characteristics, and the addition of carriers to form biofilm is a common strategy for achieving Anammox in mainstream municipal wastewater. However, relatively few studies have been conducted on the evolution of the microbial community structure and functions during Anammox biofilm formation under low-temperature and low-substrate conditions. Therefore, in this study, an Anammox sludge-biofilm system was first established under low-substrate conditions (-N = (32.6 ± 2.4) mg/L,-N = (43.9 ± 1.9) mg/L). The evolution of microbial community structure and functions during the formation of Anammox biofilm was thoroughly investigated. The results showed that Anammox biofilms could form in about 20 days under low-substrate concentrations, and the total nitrogen removal efficiency of the reaction system could be maintained at 67.1% ± 2.5%. The addition of carriers provided a diverse ecological niche for the anammox sludge and significantly increased the species richness and diversity of the sludge-biofilm system. The addition of carriers also played a selective role in the anammox bacterial community. The relative abundance of the filamentous Chloroflexi, which served as a skeleton, decreased from 20.9% in the seed sludge to 18.6%. In contrast, the relative abundances of norank_f__norank_o__norank_c__WWE3 and norank_f__AKYH767 increased significantly from 1.9% and 0 to 13.8% and 7.6%, respectively. The results of β nearest taxon index also demonstrated that the low-substrate conditions and the addition of carriers increased the role of homogeneous selection in the process of microbial community construction. In addition, the formation of biofilm enhanced the metabolic functions of Anammox communities related to cell growth, such as amino acid metabolism, translation, replication and repair, nucleotide metabolism, and cell growth and death. At the same time, it also strengthened ecological functions such as denitrification, nitrate respiration, nitrate reduction, and those related to the carbon cycle. However, metabolic functions such as energy metabolism, membrane transport and signal transduction related to information exchange, as well as nitrogen respiration, nitrite respiration, Anammox function, and ecological functions related to the sulfur cycle, were significantly reduced. Close-