Metagenomic analysis of age-dependent microbial dynamics in dual-media rapid sand filters treating groundwater

Biofiltration using rapid sand filters is an often used and highly effective method to produce drinking water from anoxic groundwater, yet the microbial processes underlying contaminant removal remain poorly understood. This study used metagenomic sequencing to analyze the microbial communities of dual-media filters of varying ages, yielding 34 high-quality and 177 medium-quality metagenome-assembled genomes (MAGs), including 102 MAGs harboring genes involved in methane, iron or manganese oxidation, and nitrification. Methanotrophic bacteria from the Methylomonadaceae family were abundant in both the youngest and oldest anthracite layers. The dominant methanotroph shifted from a MAG possessing both soluble and particulate methane monooxygenases in the two-month-old filters to MAGs only containing particulate methane monooxygenases in older filters, possibly due to changes in copper availability. Iron oxidation was restricted to the anthracite layer and linked predominantly to Gallionella, which encoded cluster 1 outer membrane cytochromes Cyc2. Gallionella MAGs also became more abundant with filter age, indicating increasing relevance of biological iron oxidation in mature filters. The nitrifying community was dominated by Nitrosomonas and Candidatus Nitrotoga in the two-month-old filter, with the latter being replaced by Nitrospira as the most abundant NOB in older filters. Canonical Nitrospira far exceeded comammox Nitrospira in abundance, except in the oldest sand layers. These results challenge the previously posed assumption that Nitrospira dominance in rapid sand filters is primarily due to comammox activity. Furthermore, it indicates that long operational times are required for comammox Nitrospira to achieve significant abundance. Intriguingly, Nitrospira MAGs contained multi-copper oxidase-type putative manganese oxidases and cluster 3 cyc2 genes. Phylogenetic analysis could not conclusively link the Nitrospira Cyc2 to iron or manganese oxidation, warranting further study of their role in the removal of these compounds. Overall, these findings reveal dynamic, age-dependent changes in microbial communities and their functions within rapid sand filters, offering new targets for physiological studies of bacteria involved in contaminant removal from anoxic groundwater during drinking water production. HIGHLIGHTSO_LIMethanotrophic community shifts between young and mature rapid sand filters. C_LIO_LICluster 1 Cyc2-encoding Gallionella mediate biological iron oxidation. C_LIO_LICandidatus Nitrotoga and Nitrosomonas dominate nitrification in young filters. C_LIO_LIComammox Nitrospira become abundant after prolonged operational periods. C_LIO_LICanonical Nitrospira, not comammox, constitute high Nitrospira abundances. C_LIO_LIAbundant cluster 3 Cyc2-containing Nitrospira might catalyze metal oxidation. C_LI