Microbiome re-assembly enhances manganese and ammonium co-removal in sand biofilters

• Activated sludge enhanced Mn(II) and NH 4 + removal processes in sand biofilters . • Low ammonium concentrations (∼ 1 mg/L) delayed the Mn removal process. • Microbial composition, not diversity, drives sand biofilters removal efficiency. • Bacteroidota is probably a biomarker for the Mn(II) removal process. • Comammox N. was crucial for nitrification during biofilter ripening’s early phase. Sand biofiltration is widely utilized to remove undesirable substances, such as divalent manganese [Mn (II)] and ammonium (NH 4 + ), from water resources. Long-term consumption of water with pollutants, notably excessive Mn(II), would result in lower neuropsychological performance and hyperactivity behaviors in children. While sand biofiltrations have been extensively inplemented for pollutant removal, their operational effeciency is contstrained by the long startup periods. This study evaluated Mn(II) and NH 4 + removal in two sand biofilters, one with NH 4 + (named NB) and one without (named MB) under different inoculum conditions, over 310 days of operation. Biofilters inoculated with sediments showed Mn(II) removal rates of 15.7 % (MB) and 9.0 % (NB), respectively, with no NH 4 + removal observed. After adding activated sludge (AS), Mn(II) removal increased to 56.3 % and 37.5 %, and NH 4 + -N removal reached 38.3 %. AS significantly improved removal efficiency and shaped microbial community structure. Linear discriminant analysis (LDA) effect size (LEfSe) analysis identified various key microorganisms, including Sphingomonas , Bradyrhizobium , Rhizobacter , Pedomicrobium , and Legionella , significantly proliferated post-AS inoculation. Spearman correlation indicated Acidobacteriota was positively correlated with Mn(II) removal, while Nitrospirota was linked to NH 4 + removal. Apart from Pedomicrobium , several unclassified species might be associated with the Mn(II) removal process. The amplicon sequencing combined RT-qPCR results indicated enhanced NH 4 + oxidation at the early stage of the biofilter ripening, attributed to the rapid enrichment of comammox Nitrospira . The study highlighted the importance of inoculum selection in achieving efficient biofilter startup.