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Biological rapid sand filtration is a commonly employed method for the removal of organic and inorganic impurities in water which relies on the degradative properties of microorganisms for the removal of diverse contaminants, but their bioremediation capabilities vary greatly across waterworks. Bioaugmentation efforts with degradation-proficient bacteria have proven difficult due to the inability of the exogenous microbes to stably colonize the sand filters. Plasmids are extrachromosomal DNA elements that can often transfer between bacteria and facilitate the flow of genetic information across microbiomes, yet their ability to spread within rapid sand filters has remained unknown. Here, we examine the permissiveness of rapid sand filter communities toward four environmentally transmissible plasmids, RP4, RSF1010, pKJK5, and TOL (pWWO), using a dual-fluorescence bioreporter platform combined with fluorescence-activated cell sorting (FACS) and 16S rRNA gene amplicon sequencing. Our results reveal that plasmids can transfer at high frequencies and across distantly related taxa from rapid sand filter communities, emphasizing their potential suitability for introducing bioremediation determinants in the microbiomes of underperforming water purification plants. IMPORTANCE The supply of clean water for human consumption is being challenged by the appearance of anthropogenic pollutants in groundwater ecosystems. Because many plasmids can transfer horizontally between members of bacterial communities, they comprise promising vectors for the dissemination of pollutant-degrading genetic determinants within water purification plants. However, their ability to spread within groundwater-fed rapid sand filters has not been explored. Here, we investigate the transfer dynamics of four transmissible plasmids across rapid sand filter communities originating from three different waterworks in Denmark. Our results revealed a significant ability of natural plasmids to transfer at high frequencies and across distantly related taxa in the absence of plasmid selection, indicating their potential suitability as vectors for the spread of bioremediation determinants in water purification plants. Future work is required to assess the biotechnological applicability and long-term maintenance of exogenous plasmids within sand filter communities.

The supply of clean water for human consumption is being challenged by the appearance of pesticide pollutants in groundwater ecosystems. Biological rapid sand filtration is a commonly employed method for the removal of organic and inorganic impurities in water which relies on the degradative properties of microorganisms for the removal of diverse contaminants, including pesticides. Although sustainable and relatively inexpensive, the bioremediation capabilities of rapid sand filters vary greatly across waterworks. Bioaugmentation efforts with degradation-proficient bacteria have proven difficult due to the inability of the exogenous microbes to stably colonize the sand filters. Pesticide degrading genes, however, are often encoded naturally by plasmids—extrachromosomal DNA elements that can transfer between bacteria—yet their ability to spread within rapid sand filters have remained unknown. To evaluate the potential use of plasmids for the dissemination of pesticide degrading genes, we examined the permissiveness of rapid sand filter communities towards four environmental transmissible plasmids; RP4, RSF1010, pKJK5 and TOL (pWWO), using a dual-fluorescent bioreporter platform combined with FACS and 16S rRNA gene amplicon sequencing. Our results reveal that plasmids can transfer at high frequencies and across distantly related taxa from rapid sand filter communities, emphasizing their suitability for introducing pesticide degrading determinants in the microbiomes of underperforming water purification plants.

Drinking water supply is in many parts of the world based on groundwater. Groundwater often contains methane, which can be oxidized by methanotrophs upon aeration. Sand from rapid sand filters fed with methane-rich groundwater can remove some pesticides (Hedegaard and Albrechtsen in Water Res 48:71-81, 2014). We enriched methanotrophs from filter sand and investigated whether they could drive the degradation of various pesticides. To enrich for methanotrophs, we designed and operated four laboratory-scale, continuously methane-fed column reactors, inoculated with filter sand and one control column fed with tap water. When enrichments were obtained, methane was continuously supplied to three reactors, while the fourth was starved for methane for 1 week, and the reactors were spiked with ten pesticides at groundwater-relevant concentrations (2.1-6.6 μg/L). Removal for most pesticides was not detected at the investigated contact time (1.37 min). However, the degradation of phenoxy acids was observed in the methanotrophic column reactor starved for methane, while it was not detected in the control column indicating the importance of methanotrophs. Phenoxy acid removal, using dichlorprop as a model compound, was further investigated in batch experiments with methanotrophic biomass collected from the enrichment reactors. Phenoxy acid removal (expressed per gram of matrix sand) was substantially improved in the methanotrophic enrichment compared to parent filter sand. The presence of methane did not clearly impact dichlorprop removal but did impact mineralization. We suggest that other heterotrophs are responsible for the first step in dichlorprop degradation, while the subsequent steps including ring-hydroxylation are driven by methanotrophs.

Summary Molecular methods to investigate functional groups in microbial communities rely on the specificity and selectivity of the primer set towards the target. Here, using rapid sand filters for drinking water production as model environment, we investigated the consistency of two commonly used quantitative PCR methods to enumerate ammonia‐oxidizing bacteria (AOB): one targeting the phylogenetic gene 16S rRNA and the other, the functional gene amoA. Cloning‐sequencing with both primer sets on DNA from two waterworks revealed contrasting images of AOB diversity. The amoA‐based approach preferentially recovered sequences belonging to Nitrosomonas Cluster 7 over Cluster 6A ones, while the 16S rRNA one yielded more diverse sequences belonging to three AOB clusters, but also a few non‐AOB sequences, suggesting broader, but partly unspecific, primer coverage. This was confirmed by an in silico coverage analysis against sequences of AOB (both isolates and high‐quality environmental sequences). The difference in primer coverage significantly impacted the estimation of AOB abundance at the waterworks with high Cluster 6A prevalence, with estimates up to 50‐fold smaller for amoA than for 16S rRNA. In contrast, both approaches performed very similarly at waterworks with high Cluster 7 prevalence. Our results highlight that caution is warranted when comparing AOB abundances obtained using different qPCR primer sets.

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 (Gulay 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.

Summary Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive and maintain the plasmids. The community permissiveness increased up to 100% in communities derived from manured soil. While the plasmid transfer frequency was significantly influenced by both the type of plasmid and the agronomic treatment, the diversity of the transconjugal pools was purely plasmid dependent and was dominated by β- and γ-Proteobacteria.

Rapid sand filtration (RSF) is an economical way to treat anoxic groundwater around the world. It consists of groundwater aeration followed by passage through a sand filter. The oxidation and removal of ferrous iron, which is commonly found in anoxic groundwaters, is often believed to be a fully physicochemical process. However, persistently low temperatures in RSF across Denmark may negatively affect the kinetics of chemical oxidation. The slower chemical oxidation of ferrous iron may increase the chances for iron bioconversion by neutrophilic iron-oxidizing bacteria (FeOB), which are found naturally in many environments. In this study, we used a combination of a cultivation-based opposing gradient enrichment technique and 16S rRNA gene targeted molecular tools to isolate, quantify and identify FeOB from a RSF. The microscopic quantification of selectively enriched FeOB cells revealed that in RSF, neutrophilic iron oxidizers were present at the level of up to 7 × 105 cells g−1 sediment. The spatial abundance and diversity of FeOB inferred by denaturing gradient gel electrophoresis fingerprinting differed greatly both between and within individual sand filters. The results suggest a larger than assumed role of FeOB in iron removal at waterworks using RSF technologies.

Here, we document microbial communities in rapid gravity filtration units, specifically serial rapid sand filters (RSFs), termed prefilters (PFs) and after- filters (AFs), fed with anoxic groundwaters low in organic carbon to prepare potable waters. A comprehensive 16S rRNA-based amplicon sequencing survey revealed a core RSF microbiome comprising few bacterial taxa (29-30 genera) dominated by Nitrospirae, Proteobacteria and Acidobacteria, with a strikingly high abundance (75-87±18%) across five examined waterworks in Denmark. Lineages within the Nitrospira genus consistently comprised the second most and most abundant fraction in PFs (27±23%) and AFs (45.2±23%), respectively, and were far more abundant than typical proteobacterial ammonium-oxidizing bacteria, suggesting a physiology beyond nitrite oxidation for Nitrospira. Within the core taxa, sequences closely related to types with ability to oxidize ammonium, nitrite, iron, manganese and methane as primary growth substrate were identified and dominated in both PFs (73.6±6%) and AFs (61.4±21%), suggesting their functional importance. Surprisingly, operational taxonomic unit richness correlated strongly and positively with sampling location in the drinking water treatment plant (from PFs to AFs), and a weaker negative correlation held for evenness. Significant spatial heterogeneity in microbial community composition was detected in both PFs and AFs, and was higher in the AFs. This is the first comprehensive documentation of microbial community diversity in RSFs treating oligotrophic groundwaters. We have identified patterns of local spatial heterogeneity and dispersal, documented surprising energy-diversity relationships, observed a large and diverse Nitrospira fraction and established a core RSF microbiome.

The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria , but also Arthrobacter sp., a gram-positive member of the Actinobacteria . The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 × 10 −2 ± 3.84 × 10 −2 . This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.

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 NH 4 + 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.

Biological rapid sand filters are often used to remove ammonium from groundwater for drinking water supply. They often operate under dynamic substrate and hydraulic loading conditions, which can lead to increased levels of ammonium and nitrite in the effluent. To determine the maximum nitrification rates and safe operating windows of rapid sand filters, a pilot scale rapid sand filter was used to test short-term increased ammonium loads, set by varying either influent ammonium concentrations or hydraulic loading rates. Ammonium and iron (flock) removal were consistent between the pilot and the full-scale filter. Nitrification rates and ammonia-oxidizing bacteria and archaea were quantified throughout the depth of the filter. The ammonium removal capacity of the filter was determined to be 3.4 g NH4–N m−3 h−1, which was 5 times greater than the average ammonium loading rate under reference operating conditions. The ammonium removal rate of the filter was determined by the ammonium loading rate, but was independent of both the flow and influent ammonium concentration individually. Ammonia-oxidizing bacteria and archaea were almost equally abundant in the filter. Both ammonium removal and ammonia-oxidizing bacteria density were strongly stratified, with the highest removal and ammonia-oxidizing bacteria densities at the top of the filter. Cell specific ammonium oxidation rates were on average 0.6 × 102 ± 0.2 × 102 fg NH4–N h−1 cell−1. Our findings indicate that these rapid sand filters can safely remove both nitrite and ammonium over a larger range of loading rates than previously assumed.

Ammonia oxidizing bacteria (AOB) perform the first step of nitrification, a key step in the Nitrogen cycle in both natural and engineered systems. In addition to their well-known role in wastewater treatment, they are also essential in rapid sand filter at waterworks treating anaerobic groundwater for drinking water production. Being able to quantify precisely the abundance of this functional group is thus important to be able monitor these processes.AOB are moderately diverse Beta-Proteobacteria that all carry the amoA gene coding for the ammonia monooxigenase. Therefore, molecular quantification can be carried out by targeting either the 16S rRNA gene or amoA, for which standard primer sets are widely used. Using these two approaches to quantify AOB abundance across three Danish rapid sand filters (RSFs) revealed a significant discrepancy: in two RSFs, the amoA-based qPCR consistently yielded estimate ~50 fold lower than that obtained with the 16S one. We carried out cloning sequencing and coverage analysis of the primer sets to explain this observation. Result showed that the primer sets have an adequate specificity but differ in their coverage. In silico analysis indicated that the amoA primer set has a narrower coverage than the 16S rRNA one and thus led to an underestimation of AOB in RSFs hosting broad AOB diversity. This highlights the importance of the choice of primer set to quantify functional groups in environmental samples.

Microbial communities derived from soils subject to different agronomic treatments were challenged with three broad host range plasmids, RP4, pIPO2tet and pRO101, via solid surface filter matings to assess their permissiveness. Approximately 1 in 10 000 soil bacterial cells could receive and maintain the plasmids. The community permissiveness increased up to 100% in communities derived from manured soil. While the plasmid transfer frequency was significantly influenced by both the type of plasmid and the agronomic treatment, the diversity of the transconjugal pools was purely plasmid dependent and was dominated by β- and γ-Proteobacteria.

There is a wealth of evidence indicating that mobile genetic elements can spread in natural microbial communities. However, little is known regarding the fraction of the community that actually engages in this behavior. Here we report on a new approach to quantify the fraction of a bacterial community that is able to receive and maintain an exogenous conjugal plasmid termed community permissiveness. Conjugal transfer of a broad-host-range plasmid labeled with a zygotically inducible green fluorescent protein (RP4:: gfp ) from a donor strain ( Pseudomonas putida ) to a soil bacterial suspension was examined. The mixture of cells was incubated on membrane filters supported by different solid media. Plasmid transfer was scored by in situ visualization of green fluorescent transconjugant microcolonies, and host range was determined by traditional plating or microcolony isolation by using a micromanipulator. Among the conditions tested, the highest plasmid transfer incidence (approximately 1 transfer per 10 4 soil bacteria) was measured after 48 h of incubation on either a 10% soil extract or a 10-fold diluted R2A medium. Stereomicroscopy combined with image analysis allowed easy examination and enumeration of green fluorescent microcolonies. In all experiments, however, stereomicroscopy consistently underestimated the number of conjugation events (approximately 10-fold) in comparison to confocal laser scanning microscopy. The plasmid host range was broad and included bacteria belonging to the Alphaproteobacteria , Betaproteobacteria , and Gammaproteobacteria classes of proteobacteria. The isolation of transconjugant microcolonies by micromanipulation greatly extended the estimated plasmid host range among soil bacteria. The new approach can be applied to examine the permissiveness of various communities toward receipt of different mobile elements.

Rapid sand filtration is an economical way to treat anoxic groundwaters and involves aeration followed by particulate and soluble substrate removal via deep bed filtration. The anoxic source groundwater can contain several potential electron donors (CH4, Fe2+, Mn2+, NH4+ and assimilable organic carbon) while oxygen (O2) is the electron acceptor provided during the aeration process. Numerous previous studies have described neutrophilic iron oxidizers as a bacterial guild with a special niche preference, especially the transition zone between aerobic and anoxic regions, where abiotic chemical oxidation of iron would be retarded. For that reason, no attempts have been documented to describe the density and diversity of iron oxidizing bacteria (FeOB) in oxic neutrophilic environments. Under low temperatures (5 to 10°C) conditions, as typically found in groundwater, extremely low rates of chemical iron oxidation (t1/2: 315min.) have been documented. This assumed slow chemical oxidation of Fe2 + in rapid sand filters may allow certain bacteria to oxidize iron concurrently with the ongoing slow chemical oxidation. Hence, we aimed to investigate the abundance, diversity, and spatial distribution of iron oxidizing bacterial in the highly oxic environments found in typical rapid sand filters.The neutrophilic FeOB were enriched by the Fe2+/O2 opposing gradient technique and quantified by MPN methodology. Diversity fingerprints of the enrichment cultures were obtained with a 16S rRNA targeted DGGE technique, and dominant bands were isolated and sequenced for identification of dominant enrichment members. Enrichment were microscopically examined via CSLM in combination with FeOB specific or generic cytostains to verify enrichments, check cell morphologies and quantify cell densities. Our results indicate that neutrophilic iron oxidizers in highly oxic environments like drinking water treatment systems can be abundant (5 E+04 to 7 E+05 cells per gram of wet sand material). It was furthermore observed that the diversity of the cultivated dominant iron oxidizers differs substantially from those typically observed in aerobic/anoxic transition zones.

Drinking water production from groundwater will often require removal of several compounds such as ammonia, manganese, ferrous iron, methane, sulphides or natural organic matter (NOM). In rapid sand filters this may be mediated through microbial processes. Sometimes the filters unfortunately fail to meet the design criteria, and a deeper insight in the underlying processes would provide a necessary platform to solve the problems. Efficient removal of potential microbial substrates is essential for production of biostable water which is required when the produced drinking water is stored and distributed without a disinfection residual as e.g. in Denmark. We have developed a toolbox including investigations of the presence of required microorganisms. To investigate the presence and density of various microbial fractions, qPCR-methods were established for quantification of ammonium oxidizing (AOB, AOA), nitrite oxidizing (NOB), iron oxidizing (IOB) and methane oxidizing (MeOB) microorganisms. In addition, pyrosequencing of the full microbiome of the sand filters revealed a high diversity, and especially the presence of a very large and dominating population of Nitrospira was surprizing. Nitrification was particularly investigated in full scale filters, and lab-scale CST-columns incubated with depth specific samples of filter material allowed for investigation of the depth specific kinetics and maximum removal capacity. Additionally, pilot scale column experiments allowed for investigation of e.g. increased load of ammonium due to increased hydraulic load versus increased concentration, physical space in the filter material and its surface qualities. A safe operational windows in terms of load was identified during short term up-shifts in the ammonium load to the different columns. This showed the importance of the total load no matter the increase was due to hydraulic load or concentration. Based on the obtained insight the functionality of the filters could be optimized. Addition of limiting micronutrients such as phosphorous or cupper (which specifically stimulating nitrification since cupper is an essential metal in the ammonium mono oxygenase) was able to increase nitrification rate, to overcome incomplete nitrification with accumulation of nitrite, and to reduce the startup time of the microbial processes in new filters. This presentation provides an overview of a number of research projects on biological rapid sand filters conducted during the last 5 years.

Rapid sand filtration is a traditional and common technology for drinking water purification from groundwater. Despite its wide scale and long-term use, the diversity and characterization of microbial communities in these engineered systems have remained unexplored and their roles in removal performances yet to be discovered. In order to explore the microbial ecology of these systems, we conducted 16S rRNA gene (rDNA) based 454 pyrosequencing as a deep sequencing approach to 94 sample cores retrieved from 5 different waterworks including proper biological replication. This comprehensive sampling of replicate rapid sand filters across many waterworks together with high-throughput sequencing provides a first glimpse into the microbial communities in rapid sand filters and their potential roles in the treatment process.

Rapid sand filters used in groundwater treatment remove ammonium, iron and manganese from the water. Ammonium is removed biologically by nitrifying microorganisms attached on the sand surface. Nitrification kinetics and activity is strongly affected by filter design and operation, which are the key parameters in process optimization. Nitrification optimization needs a detailed insight of the process and the way it takes place in the filter. Filters are often considered in a “black box” approach, where data are only available for influent and effluent and the entire filter is assumed homogenous. The aim of this study is to investigate nitrification activity in a rapid sand filter, with focus on its homogeneity and how it relates to filter performance. Two groundwater treatment plants in Denmark were selected for the experimental investigations. Plant 1 operates a single line of pre and after filters and has been well performing over the last years. Plant 2 consists of two separate lines, each one with pre and after filtration steps. Plant 2 has experienced challenges in removing ammonium below the 0.05 mg/L regulatory limit especially in one of the two lines. Sand core samples were taken from the after filter in Plant 1 and the after filters in both lines of plant 2. Core samples were divided according to depth and nitrification activity was measured in a lab scale assay. The method consists in a continuous flow mini-column where influent and effluent are monitored for all nitrogen species. Kinetics and maximum nitrification capacity are derived and used to quantify nitrification activity.Nitrification activity was concentrated at the top 10 cm of filter depth, and maximum nitrification capacity was 7 g NH4+-N/ m3 sand/h compared with 0.8-0.4 g NH4+-N/ m3 sand/h in the middle and bottom layers. A water sampler was installed in the full scale filter of plant 1 to observe the ammonium profile with depth. Ammonium was removed within the upper 15 cm with a removal rate ranging of 3.6- 7.7 g NH4+-N/ m3 sand/h. Full scale observations fit with the lab scale activity measurements showing that the upper layer of the filter is where nitrification mostly happens. Deeper layers that are less active, provide extra nitrifying capacity in case ammonium is not removed within the top 15 cm. qPCR counts for ammonium oxidizing bacteria showed a decrease from 5*107 cells /gr sand at the top of the filter to 2*105 cells /gr sand in the lowest 20 cm. From this study results that rapid sand filters are not homogenous in terms of biological activity. This can be an important consideration when modeling these units and as a basis for process optimization.