Density and distribution of nitrifying guilds in rapid sand filters for drinking water production: Dominance of Nitrospira spp

We investigated the density and distribution of total bacteria, canonical Ammonia Oxidizing Bacteria (AOB) (Nitrosomonas plus Nitrosospira), Ammonia Oxidizing Archaea (AOA), as well as Nitrobacter and Nitrospira in rapid sand filters used for groundwater treatment. To investigate the spatial distribution of these guilds, filter material was sampled at four drinking water treatment plants (DWTPs) in parallel filters of the pre- and after-filtration stages at different locations and depths. The target guilds were quantified by qPCR targeting 16S rRNA and amoA genes. Total bacterial densities (ignoring 16S rRNA gene copy number variation) were high and ranged from 10⁹ to 10¹⁰ per gram (10¹⁵ to 10¹⁶ per m³) of filter material. All examined guilds, except AOA, were stratified at only one of the four DWTPs. Densities varied spatially within filter (intra-filter variation) at two of the DWTPs and in parallel filters (inter-filter variation) at one of the DWTPs. Variation analysis revealed random sampling as the most efficient strategy to yield accurate mean density estimates, with collection of at least 7 samples suggested to obtain an acceptable (below half order of magnitude) density precision. Nitrospira was consistently the most dominant guild (5-10% of total community), and was generally up to 4 orders of magnitude more abundant than Nitrobacter and up to 2 orders of magnitude more abundant than canonical AOBs. These results, supplemented with further analysis of the previously reported diversity of Nitrospira in the studied DWTPs based on 16S rRNA and nxrB gene phylogeny (Gülay et al., 2016; Palomo et al., 2016), indicate that the high Nitrospira abundance is due to their comammox (complete ammonia oxidation) physiology. AOA densities were lower than AOB densities, except in the highly stratified filters, where they were of similar abundance. In conclusion, rapid sand filters are microbially dense, with varying degrees of spatial heterogeneity, which requires replicate sampling for a sufficiently precise determination of total microbial community and specific population densities. A consistently high Nitrospira to bacterial and archaeal AOB density ratio suggests that non-canonical pathways for nitrification may dominate the examined RSFs.