DNA- and RNA-SIP Reveal Nitrospira spp. as Key Drivers of Nitrification in Groundwater-Fed Biofilters.

Nitrification, the oxidative process converting ammonia to nitrite and nitrate, is driven by microbes and plays a central role in the global nitrogen cycle. Our earlier investigations based on 16S rRNA and amoA amplicon analysis, amoA quantitative PCR and metagenomics of groundwater-fed biofilters indicated a consistently high abundance of comammox Nitrospira Here, we hypothesized that these nonclassical nitrifiers drive ammonia-N oxidation. Hence, we used DNA and RNA stable isotope probing (SIP) coupled with 16S rRNA amplicon sequencing to identify the active members in the biofilter community when subjected to a continuous supply of NH4+ or NO2- in the presence of 13C-HCO3- (labeled) or 12C-HCO3- (unlabeled). Allylthiourea (ATU) and sodium chlorate were added to inhibit autotrophic ammonia- and nitrite-oxidizing bacteria, respectively. Our results confirmed that lineage II Nitrospira dominated ammonia oxidation in the biofilter community. A total of 78 (8 by RNA-SIP and 70 by DNA-SIP) and 96 (25 by RNA-SIP and 71 by DNA-SIP) Nitrospira phylotypes (at 99% 16S rRNA sequence similarity) were identified as complete ammonia- and nitrite-oxidizing, respectively. We also detected significant HCO3- uptake by Acidobacteria subgroup10, Pedomicrobium, Rhizobacter, and Acidovorax under conditions that favored ammonia oxidation. Canonical Nitrospira alone drove nitrite oxidation in the biofilter community, and activity of archaeal ammonia-oxidizing taxa was not detected in the SIP fractions. This study provides the first in situ evidence of ammonia oxidation by comammox Nitrospira in an ecologically relevant complex microbiome.IMPORTANCE With this study we provide the first in situ evidence of ecologically relevant ammonia oxidation by comammox Nitrospira in a complex microbiome and document an unexpectedly high H13CO3- uptake and growth of proteobacterial and acidobacterial taxa under ammonia selectivity. This finding raises the question of whether comammox Nitrospira is an equally important ammonia oxidizer in other environments.

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.

Internal porosity of mineral coating supports microbial activity in rapid sand filters for groundwater treatment.

A mineral coating develops on the filter grain surface when groundwater is treated via rapid sand filtration in drinking water production. The coating changes the physical and chemical properties of the filter material, but little is known about its effect on the activity, colonization, diversity, and abundance of microbiota. This study reveals that a mineral coating can positively affect the colonization and activity of microbial communities in rapid sand filters. To understand this effect, we investigated the abundance, spatial distribution, colonization, and diversity of all and of nitrifying prokaryotes in filter material with various degrees of mineral coating. We also examined the physical and chemical characteristics of the mineral coating. The amount of mineral coating correlated positively with the internal porosity, the packed bulk density, and the biologically available surface area of the filter material. The volumetric NH4 (+) removal rate also increased with the degree of mineral coating. Consistently, bacterial 16S rRNA and amoA abundances positively correlated with increased mineral coating levels. Microbial colonization could be visualized mainly within the outer periphery (60.6 ± 35.6 µm) of the mineral coating, which had a thickness of up to 600 ± 51 µm. Environmental scanning electron microscopic (E-SEM) observations suggested an extracellular polymeric substance-rich matrix and submicron-sized bacterial cells. Nitrifier diversity profiles were similar irrespective of the degree of mineral coating, as indicated by pyrosequencing analysis. Overall, our results demonstrate that mineral coating positively affects microbial colonization and activity in rapid sand filters, most likely due to increased volumetric cell abundances facilitated by the large surface area of internal mineral porosity accessible for microbial colonization.