van Kessel, M. A. | bioRxiv Science

bioRxiv2024

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

microbiology↗

bioRxiv2016

AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammox Nitrospira in the environment

Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be catalyzed by the concerted activity of ammonia and nitrite-oxidizing microorganisms. Only recently, complete ammonia oxidizers ( comammox), which oxidize ammonia to nitrate on their own, were identified in the bacterial genus Nitrospira, previously assumed to contain only canonical nitrite oxidizers. Nitrospira are widespread in nature, but for assessments of the distribution and functional importance of comammox Nitrospira in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite-oxidizing Nitrospira are required. Here we developed new PCR primer sets that specifically target the amoA genes coding for subunit A of the distinct ammonia monooxygenase of comammox Nitrospira. While existing primers capture only a fraction of the known comammox amoA diversity, the new primer sets cover as much as 95% of the comammox amoA clade A and 92% of the clade B sequences in a reference database containing 326 comammox amoA genes with sequence information at the primer binding sites. Application of the primers to 13 samples from engineered systems (a groundwater well, drinking water treatment and wastewater treatment plants) and other habitats (rice paddy and forest soils, rice rhizosphere, brackish lake sediment and freshwater biofilm) detected comammox Nitrospira in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox amoA was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox amoA genes. Quantitative PCR with these equimolar primer mixtures was highly sensitive and specific, and enabled the efficient quantification of clade A and clade B comammox amoA gene copy numbers in environmental samples. The measured relative abundances of comammox Nitrospira, compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox amoA-targeted primers will enable more encompassing studies of nitrifying microorganisms in diverse ecosystems.

microbiology↗