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Many factors affect the biodegradation kinetics of chemicals in test systems and the environment. Empirical knowledge is needed on how much test temperature, inoculum, test substances and co-substrates influence the biodegradation kinetics and microbial composition in the test. Water was sampled from the Gudenaa river in winter (2.7 °C) and summer (17 °C) (microbial inoculum) and combined with an aqueous stock solution of >40 petroleum hydrocarbons prepared by passive dosing. This resulted in low-concentration test systems that were incubated for 30 days at 2.7, 12 and 20 °C. Primary biodegradation kinetics, based on substrate depletion relative to abiotic controls, were determined with automated Solid Phase Microextraction coupled to GC/MS. Biodegradation kinetics were remarkably similar for summer and winter inocula when tested at the same temperature, except when cooling summer inoculum to 2.7 °C which delayed degradation relative to winter inoculum. Amplicon sequencing was applied to determine shifts in the microbial composition between season and during incubations: (1) the microbial composition of summer and winter inocula were remarkably similar, (2) the incubation and the incubation temperature had both a clear impact on the microbial composition and (3) the effect of adding >40 petroleum hydrocarbons at low test concentrations was limited but resulted in some proliferation of the known petroleum hydrocarbon degraders Nevskia and Sulfuritalea. Overall, biodegradation kinetics and its temperature dependency were very similar for winter and summer inoculum, whereas the microbial composition was more affected by incubation and test temperature compared to the addition of test chemicals at low concentrations.

Deterministic and stochastic processes are believed to play a combined role in microbial community assembly, though little is known about the factors determining their relative importance. We investigated the effect of biofilm thickness on community assembly in nitrifying moving bed biofilm reactors using biofilm carriers where maximum biofilm thickness is controlled. We examined the contribution of stochastic and deterministic processes to biofilm assembly in a steady state system using neutral community modelling and community diversity analysis with a null-modelling approach. Our results indicate that the formation of biofilms results in habitat filtration, causing selection for phylogenetically closely related community members, resulting in a substantial enrichment of Nitrospira spp. in the biofilm communities. Stochastic assembly processes were more prevalent in biofilms of 200 µm and thicker, while stronger selection in thinner (50 µm) biofilms could be driven by hydrodynamic and shear forces at the biofilm surface. Thicker biofilms exhibited greater phylogenetic beta-diversity, which may be driven by a variable selection regime caused by variation in environmental conditions between replicate carrier communities, or by drift combined with low migration rates resulting in stochastic historical contingency during community establishment. Our results indicate that assembly processes vary with biofilm thickness, contributing to our understanding of biofilm ecology and potentially paving the way towards strategies for microbial community management in biofilm systems.

Current in the milliampere range can be used for electrochemical inactivation of bacteria. Yet, bacteria-including antibiotic resistant bacteria (ARB)-may be subjected to sub-lethal conditions due to imperfect mixing or energy savings measures during electrochemical disinfection. It is not known whether such sublethal current intensities have the potential to stimulate plasmid transfer from ARB. In this study, conjugal transfer of plasmid pKJK5 was investigated between Pseudomonas putida strains under conditions reflecting electrochemical disinfection. Although the abundance of culturable and membrane-intact donor and recipient cells decreased with applied current (0–60 mA), both transconjugant density and transconjugant frequency increased. Both active chlorine and superoxide radicals were generated electrolytically, and ROS generation was induced. In addition, we detected significant over expression of a core oxidative stress defense gene (ahpCF) with current. Expression of selected conjugation related genes (traE, traI, trbJ, and trbL) also significantly correlated with current intensity. A link between ROS accumulation, SOS response and enhanced conjugation is therefore the plausible consequence of sublethal current exposure. These findings suggest that sub-lethal intensities of current can enhance conjugal plasmid transfer, and that it is essential that conditions of electrochemical disinfection (applied voltage, current density, time and mixing) be carefully controlled to avoid conjugal ARG transmission.

Biodegradation in the aquatic environment occurs in the presence of many chemicals, while standard simulation biodegradation tests are conducted with single chemicals. This study aimed to investigate the effect of the presence of additional chemicals on (1) biodegradation kinetics of individual chemicals and (2) the microbial composition in test systems. Parallel mixture and single substance experiments were conducted for 9 chemicals (phenethyl benzoate, oxacycloheptadec-10-en-2-one, α-ionone, methyl 2-naphthyl ether, decan-5-olide, octan-2-one, 2'-acetonaphthanone, methyl N-methylanthranilate, (+)-menthone) using inoculum from a Danish stream. Biotic and abiotic test systems were incubated at 12 °C for 1-30 days. Primary biodegradation kinetics were then determined from biotic/abiotic peak area ratios using SPME GC/MS analysis. The effect of the mixture on biodegradation varied with test chemical and was more pronounced for chemicals with lag-phases above 14 days: two chemicals degraded in the mixture but not when tested alone (i.e., positive mixture effect), and two degraded when tested alone but not in the mixture (i.e., negative mixture effect). Microbial composition (16S rRNA gene amplicon sequencing) was highly affected by 14 days incubation and the presence of the mixture (significant carbon source), but less by single chemicals (low carbon source). Growth on chemical mixtures resulted in consistent proliferation of Pseudomonas and Malikia, while specific chemicals increased the abundance of putative degraders belonging to Novosphingobium and Zoogloea. The chemical and microbiological results support (1) that simulation biodegradation kinetics should be determined in mixtures at low environmentally relevant concentrations and (2) that degradation times beyond some weeks are associated with more uncertainty.

Wastewater Treatment Plants (WWTPs) contain a diverse microbial community with high cell density. They constantly receive antimicrobial residues and resistant strains and, therefore, may offer conditions for the Horizontal Gene Transfer (HGT) of antimicrobial resistance determinants, transmitting clinically important genes between, e.g., enteric and environmental bacteria and vice versa. Despite the clinical importance, tools for predicting HGT are still under-developed. In this study, we examined to which extent microbial community composition, as inferred by partial 16S rRNA gene sequences, can predict plasmid permissiveness, i.e., the ability of cells to receive a plasmid through conjugation, for microbial communities in the water cycle, using data from standardized filter mating assays using fluorescent bio-reporter plasmids. We leveraged a range of machine learning models for predicting the permissiveness for each taxon in the community, translating to the range of hosts a plasmid is able to transfer to, for three broad host-range resistance plasmids (pKJK5, pB10, and RP4). Our results indicate that the predicted permissiveness from the best performing model (random forest) showed a moderate-to-strong average correlation of 0.45 for pB10 (95% CI: 0.42-0.52), 0.42 for pKJK5 (0.95% CI: 0.38-0.45) and 0.52 for RP4 (0.95% CI:0.45-0.55) with the experimental permissiveness in the unseen test dataset. Predictive phylogenetic signals occurred despite these being broad host-range plasmids. Our results provide a framework that contributes to assessing the risk of AMR pollution in wastewater systems. The predictive tool is available as a an application under https://github.com/DaneshMoradigaravand/PlasmidPerm.

Biodegradation of organic chemicals emitted to the environment is carried out by mixed microbial communities growing on multiple natural and xenobiotic substrates at low concentrations. This study aims to (1) perform simulation type biodegradation tests at a wide range of mixture concentrations, (2) determine the concentration effect on the biodegradation kinetics of individual chemicals, and (3) link the mixture concentration and degradation to microbial community dynamics. Two hundred ninety-four parallel test systems were prepared using wastewater treatment plant effluent as inoculum and passive dosing to add a mixture of 19 chemicals at 6 initial concentration levels (ng/L to mg/L). After 1-30 days of incubation at 12 °C, abiotic and biotic test systems were analyzed using arrow solid phase microextraction and GC-MS/MS. Biodegradation kinetics at the highest test concentrations were delayed for several test substances but enhanced for the reference chemical naphthalene. Test concentration thus shifted the order in which chemicals were degraded. 16S rRNA gene amplicon sequencing indicated that the highest test concentration (17 mg C/L added) supported the growth of the genera Acidovorax, Novosphingobium, and Hydrogenophaga, whereas no such effect was observed at lower concentrations. The chemical and microbial results confirm that too high mixture concentrations should be avoided when aiming at determining environmentally relevant biodegradation data.

High rate activated sludge (HRAS) systems redirect organics into highly biodegradable sludge and nutrients into microbial proteins. This study evaluates anoxic HRAS for nitrogen and carbon recovery. The reactor treated synthetic wastewater at solids retention times (SRTs) of 5, 3 and 1 days. Denitrification rates varied between 0.15 and 0.19 g-NO3-N g-TSS-1 d-1 (total suspended solids per day) and all conditions showed favourable settling. The highest sludge yield, obtained at SRT 1 d, was 0.75 g-TSS g-COD-1removed, double that observed for aerobic HRAS. The highest methane yield (322 mL-CH4 g-VS-1sludge) was obtained from sludge wasted at 3 d SRT. Both 1 d and 3 d SRTs showed favourable energy recovery, with 14 % of the organics recovered as methane. All conditions yielded sludge with protein content ranging between 24 and 27 % of dry weight and similar amino acid profile, comparable to traditional proteins. Thus, denitrifying HRAS recovers resources as its aerobic counterpart, allowing for nitrogen removal via denitrification, more stable compared to mainstream partial nitritation anammox typically combined with aerobic HRAS.

Plasmid-specific bacteriophages specifically infect bacteria carrying conjugal plasmids. While wastewater has been used as isolation source for such phages, to date, only the distribution and ecology of RNA phages specific to the F plasmid have been described, because they serve as a water quality indicator. Yet, several other plasmid classes have higher clinical and ecological relevance, and the distribution, fate, and ecology of the phages that target them remain uncharacterized. We aimed to (i) provide an experimental platform to quantify the abundance of plasmid-specific phages applicable to several different conjugal plasmid classes, (ii) describe the distribution of such phages in wastewater systems, and (iii) relate their abundance to plasmid abundance and to municipal wastewater treatment processes. We introduced four model conjugal plasmids, belonging to incompatibility groups IncP-1, IncN, IncHI1, or IncF into an avirulent Salmonella enterica strain, for which somatic phages are at low abundance in wastewater. These strains were used in double layer agar assays with waters from contrasting sources. Plasmid-specific phages were common in wastewater but rare in river water. Hospital wastewater contained significantly more IncP-1-, but fewer IncF- and IncN- specific phages than domestic wastewater. This pattern did not match that of plasmid abundance estimated by Inc group targeting high-throughput quantitative PCR. The comparison between influent and effluent of wastewater treatment plants revealed a reduction in phage concentration by ca. 2 log, without significant contribution of primary settling. Overall, the ubiquity of these phages hints at their importance for plasmid ecology, and can provide opportunities in water quality monitoring and in ecological management of mobile resistance genes. Plasmid-specific bacteriophages specifically infect bacteria carrying conjugal plasmids. While wastewater has been used as isolation source for such phages, to date, only the distribution and ecology of RNA phages specific to the F plasmid have been described, because they serve as a water quality indicator. Yet, several other plasmid classes have higher clinical and ecological relevance, and the distribution, fate, and ecology of the phages that target them remain uncharacterized. We aimed to (i) provide an experimental platform to quantify the abundance of plasmid-specific phages applicable to several different conjugal plasmid classes, (ii) describe the distribution of such phages in wastewater systems, and (iii) relate their abundance to plasmid abundance and to municipal wastewater treatment processes. We introduced four model conjugal plasmids, belonging to incompatibility groups IncP-1, IncN, IncHI1, or IncF into an avirulent Salmonella enterica strain, for which somatic phages are at low abundance in wastewater. These strains were used in double layer agar assays with waters from contrasting sources. Plasmid-specific phages were common in wastewater but rare in river water. Hospital wastewater contained significantly more IncP-1-, but fewer IncF- and IncN- specific phages than domestic wastewater. This pattern did not match that of plasmid abundance estimated by Inc group targeting high-throughput quantitative PCR. The comparison between influent and effluent of wastewater treatment plants revealed a reduction in phage concentration by ca. 2 log, without significant contribution of primary settling. Overall, the ubiquity of these phages hints at their importance for plasmid ecology, and can provide opportunities in water quality monitoring and in ecological management of mobile resistance genes.

Horizontal gene transfer via plasmids is important for the dissemination of antibiotic resistance genes among medically relevant pathogens. Specifically, the transfer of IncHI1A plasmids is believed to facilitate the spread of antibiotic resistance genes, such as carbapenemases, within the clinically important family Enterobacteriaceae. The microbial community of urban wastewater treatment plants has been shown to be highly permissive towards conjugal transfer of IncP1 plasmids. Here, we tracked the transfer of the P1 plasmid pB10 and the clinically relevant HI1A plasmid R27 in the microbial communities present in urban residential sewage entering full-scale wastewater treatment plants. We found that both plasmids readily transferred to these communities and that strains in the sewage were able to further disseminate them. Furthermore, that R27 has a broad potential host range, but a low host divergence. Interestingly, although the majority of R27 transfer events were to members of Enterobacteriaceae, we found a subset of transfer to other families, even other phyla. Indicating, that HI1A plasmids facilitate horizontal gene transfer both within Enterobacteriaceae, but also across families of especially Gammaproteobacteria, such as Moraxellaceae, Pseudomonadaceae and Shewanellaceae. pB10 displayed a similar potential host range as R27. In contrast to R27, pB10 had a high host divergence. By culture enrichment of the transconjugant communities, we show that sewage strains of Enterobacteriaceae and Aeromonadaceae can stably maintain R27 and pB10, respectively. Our results suggest that dissemination in the urban residual water system of HI1A plasmids may result in an accelerated acquisition of antibiotic resistance genes among pathogens.

Horizontal gene transfer via plasmids is important for the dissemination of antibiotic resistance genes among medically relevant pathogens. Specifically, the transfer of IncHI1A plasmids is believed to facilitate the spread of antibiotic resistance genes, such as carbapenemases, within the clinically important family Enterobacteriaceae. The microbial community of urban wastewater treatment plants has been shown to be highly permissive towards conjugal transfer of IncP1 plasmids. Here, we tracked the transfer of the P1 plasmid pB10 and the clinically relevant HI1A plasmid R27 in the microbial communities present in urban residential sewage entering full-scale wastewater treatment plants. We found that both plasmids readily transferred to these communities and that strains in the sewage were able to further disseminate them. Furthermore, that R27 has a broad potential host range, but a low host divergence. Interestingly, although the majority of R27 transfer events were to members of Enterobacteriaceae, we found a subset of transfer to other families, even other phyla. Indicating, that HI1A plasmids facilitate horizontal gene transfer both within Enterobacteriaceae, but also across families of especially Gammaproteobacteria, such as Moraxellaceae, Pseudomonadaceae and Shewanellaceae. pB10 displayed a similar potential host range as R27. In contrast to R27, pB10 had a high host divergence. By cultivative enrichment of the transconjugant communities, we show that sewage strains of Enterobacteriaceae and Aeromonadaceae can stably maintain R27 and pB10, respectively. Our results suggest that dissemination in the urban residual water system of HI1A plasmids may result in an accelerated acquisition of antibiotic resistance genes among pathogens.

Bacterial conjugative plasmids could carry genes involved in pesticide biodegradation. However, its conjugal transfer is affected by stressors such as metals. Our aim was to determine if bacterial cells exposed to copper can transfer by conjugation the catabolic plasmid pRO101 and assessed the expression of traK gene. Cupriavidus pinatubonensis AEO106 and Pseudomonas putida KT2440 were used as donor and recipient strains, respectively. Mating assays were carried out in presence of Cu2+ (0.3, 0.8, 1.6, 3.9 or 7.9 mM) or H2O2 (0.1, 0.2, 0.5, 1, 2 or 5 mM). The expression of traK gene and the effect of Cu2+ on donor cells was determined by RTqPCR and flow cytometry, respectively. Cytoplasmic membrane of 90% of donor cells was damaged by Cu2+, but the transference of pRO101 plasmid occurred at a proportion similar to the control. Moreover, traK gene expression was reduced (c.a. 50%) by Cu2+. This demonstrated that damaged donor cells were still able to transfer the pRO101 plasmid, allowing a rescue of these genetic material and contribute to self-purifying processes in the environment. Although without ruling out the possibility that transformation occurred, the expression of the traK gene suggested that conjugation occurred between damaged donor cells and recipient cells.

Biodegradation kinetics data are keystone for evaluating the environmental persistence and risk of chemicals. Biodegradation kinetics depend highly on the prevailing temperature, which influences microbial community structures, metabolic rates, and chemical availability. There is a lack of high-quality comparative biodegradation kinetics data that are determined at different test temperatures but with the same microbial inoculum and chemical availability. The present study was designed to determine the effect of test temperature on the biodegradation kinetics of hydrocarbons while avoiding confounding factors. We used inocula from a Northern river (2.7 °C) and a Central European river (12.5 °C). Aqueous stock solutions containing 45 individual hydrocarbons were generated by passive dosing and added to river water containing the native microorganisms. Compound-specific biodegradation kinetics were then determined at 2.7, 12, and 20 °C based on substrate depletion. Main findings comprise the following: (1) Degradation half-times (DegT50) of 34 test chemicals were determined at different test temperatures and were largely consistent with the Arrhenius equation (activation energy, 65.4 kJ/mol). (2) Differences in biodegradation kinetics between tested isomers were rather limited. (3) The recent lowering of standard test temperature from 20 to 12 °C results typically in a doubling of DegT50 values and can lead to a stricter persistency assessment.

Context. Plasmid-specific bacteriophages are viruses that specifically infect bacteria carrying conjugal plasmids, typically using the conjugal pilus as receptor. As pili differ across plasmid classes, the infection range of plasmid specific phages has specificity. Wastewater has been used as isolation source for such phages, however only the distribution and ecology of F-specific RNA phages have been described to date because it is a water quality parameter (as proxy for RNA-viruses from fecal sources).Gap. The source, fate, and abundance of plasmid-specific phages are largely uncharacterized and there is a lack of experimental platforms to study them.Aim. We aimed to (i) provide an experimental platform to quantify the abundance of plasmid-specific phages for several conjugal plasmid classes and (ii) describe the distribution of such phages in wastewater systems and (iii) identify what controls their distribution.Methods. We introduced four model plasmids, belonging to different plasmid incompatibility groups (IncP1, IncN, IncH, IncF) into an avirulent Salmonella enterica serovar Typhimurium, which displays a low background abundance of somatic phages in wastewater. These strains were used in double layer agar assays with water samples from contrasting sources (hospital wastewater, influent and effluent of municipal wastewater treatment plants in Sweden and Denmark).Findings. Depending on the location and plasmid class, phage abundance ranged from below detection limit to about 5 103 pfu/mL. Hospital wastewater contained significantly more IncP-, but less IncF-, specific phages than domestic wastewaters, potentially reflecting differences in plasmid loads. The comparison between influent and effluent of WWTP revealed a reduction in phage density by 1.5 log, but no significant removal from primary clarification.Utilization. First, fate data for viruses in wastewater can inform wastewater-based epidemiology. Second, these data help understand phage-plasmid interactions, especially relevant as plasmids can disseminate AMR, and may suggest novel ways to control AMR in wastewater systems.

Urban wastewater systems (UWSs) are a main receptacle of excreted antibiotic resistance genes (ARGs) and their host microorganisms. However, we lack integrated and quantitative observations of the occurrence of ARGs in the UWS to characterize the sources and identify processes that contribute to their fate. We sampled the UWSs from three medium-size cities in Denmark, Spain, and the United Kingdom and quantified 70 clinically important extended-spectrum β-lactamase and carbapenemase genes along with the mobile genetic elements and microbial communities. Results from all three countries showed that sewage-especially from hospitals-carried substantial loads of ARGs (106-107 copies per person equivalent), but these loads progressively declined along sewers and through sewage treatment plants, resulting in minimal emissions (101-104 copies per person equivalent). Removal was primarily during sewage conveyance (65 ± 36%) rather than within sewage treatment (34 ± 23%). The extended-spectrum β-lactamase and carbapenemase genes were clustered in groups based on their persistence in the UWS compartments. The less-persistent groups were associated to putative host taxa (especially Enterobacteriaceae and Moraxellaceae), while the more persistent groups appeared horizontally transferred and correlated significantly with total cell numbers and mobile genetic elements. This documentation of a substantial ARG reduction during sewage conveyance provides opportunities for antibiotic resistance management and a caution for sewage-based antibiotic resistance surveillance.

Integrated and quantitative observations of antibiotic resistance genes (ARGs) in urban water systems (UWSs) are lacking. We sampled three UWSs for clinically important extended spectrum β-lactamase (ESBL) and carbapenemase (CP) genes, mobile genetic elements and microbial communities. Sewage – especially from hospitals – carried substantial loads of ESBL and CP genes (106– 107per person equivalent), but those loads progressively declined along the UWS, resulting in minimal emissions (101– 104copies per person equivalent). Removal was primarily during sewage conveyance (65% ± 36%) rather than within sewage treatment (34% ± 23%). The ARGs clustered in groups based on their persistence; less persistent groups were associated to putative host taxa (especiallyEnterobacteriaceaeandMoraxellaceae), while more persistent groups appear horizontally transferred as they correlated with mobile genetic elements. This first documentation of a substantial ARG reduction during sewage conveyance provides opportunities for antibiotic resistance management and a caution for sewage-based ARG surveillance.

Microbes commonly exist in diverse and complex communities where species interact, and their genomic repertoires evolve over time. Our understanding of species interaction and evolution has increased during the last decades, but most studies of evolutionary dynamics are based on single species in isolation or in experimental systems composed of few interacting species. Here, we use the microbial ecosystem found in groundwater-fed sand filter as a model to avoid this limitation. In these open systems, diverse microbial communities experience relatively stable conditions, and the coupling between chemical and biological processes is generally well defined. Metagenomic analysis of 12 sand filters communities revealed systematic co-occurrence of at least five comammox Nitrospira species, likely promoted by low ammonium concentrations. These Nitrospira species showed intrapopulation sequence diversity, although possible clonal expansion was detected in a few abundant local comammox populations. Nitrospira species showed low homologous recombination and strong purifying selection, the latter process being especially strong in genes essential in energy metabolism. Positive selection was detected for genes related to resistance to foreign DNA and phages. We found that, compared to other habitats, groundwater-fed sand filters impose strong purifying selection and low recombination on comammox Nitrospira populations. These results suggest that evolutionary processes are more affected by habitat type than by species identity. Together, this study improves our understanding of species interaction and evolution in complex microbial communities and sheds light on the environmental dependency of evolutionary processes. IMPORTANCE: Microbial species interact with each other and their environment (ecological processes) and undergo changes in their genomic repertoire over time (evolutionary processes). How these two classes of processes interact is largely unknown, especially for complex communities, as most studies of microbial evolutionary dynamics consider single species in isolation or a few interacting species in simplified experimental systems. In this study, these limitations are circumvented by examining the microbial communities found in stable and well-described groundwater-fed sand filters. Combining metagenomics and strain-level analyses, we identified the microbial interactions and evolutionary processes affecting comammox Nitrospira, a recently discovered bacterial type capable of performing the whole nitrification process. We found that abundant and co-occurrent Nitrospira populations in groundwater-fed sand filters are characterized by low recombination and strong purifying selection. In addition, by comparing these observations with those obtained from Nitrospira species inhabiting other environments, we revealed that evolutionary processes are more affected by habitat type than by species identity.

Species specific nitrogen-to-phosphorus molar ratio (NPR) has been suggested for green microalgae. Algae can store nitrogen and phosphorus, suggesting that the optimum feed concentration dynamically changes as function of the nutrient storage. We assessed the effect of varying influent NPR on microalgal cultivation in terms of microbial community stability, effluent quality and biokinetics. Mixed green microalgae (Chlorella sorokiniana and Scenedesmus sp.) and a monoculture of Chlorella sp. were cultivated in continuous laboratory-scale reactors treating used water. An innovative image analysis tool, developed in this study, was used to track microbial community changes. Diatoms proliferated as influent NPR decreased, and were outcompeted once cultivation conditions were restored to the optimal NPR range. Low NPR operation resulted in decrease in phosphorus removal, biomass concentration and effluent nitrogen concentration. ASM-A kinetic model simulation results agreed well with operational data in the absence of diatoms. The failure to predict operational data in the presence of diatoms suggest differences in microbial activity that can significantly influence nutrient recovery in photobioreactors (PBR). No contamination occurred during Chlorella sp. monoculture cultivation with varying NPRs. Low NPR operation resulted in decrease in biomass concentration, effluent nitrogen concentration and nitrogen quota. The ASM-A model was calibrated for the monoculture and the simulations could predict the experimental data in continuous operation using a single parameter subset, suggesting stable biokinetics under the different NPR conditions. Results show that controlling the influent NPR is effective to maintain the algal community composition in PBR, thereby ensuring effective nutrients uptake.

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.

Wastewater treatment plants (WWTPs) have been suggested as reservoirs and sources of antibiotic resistance genes (ARGs) in the environment. In a WWTP ecosystem, human enteric and environmental bacteria are mixed and exposed to pharmaceutical residues, potentially favoring genetic exchange and thus ARG transmission. However, the contribution of microbial communities in WWTPs to ARG dissemination remains poorly understood. Here, we examined for the first time plasmid permissiveness of an activated sludge microbial community by utilizing an established fluorescent bioreporter system. The activated sludge microbial community was challenged in standardized filter matings with one of three multidrug resistance plasmids (pKJK5, pB10, and RP4) harbored by Escherichia coli or Pseudomonas putida. Different donor–plasmid combinations had distinct transfer frequencies, ranging from 3 to 50 conjugation events per 100000 cells of the WWTP microbial community. In addition, transfer was observed to a broad phylogenetic range of 13 bacterial phyla with several taxa containing potentially pathogenic species. Preferential transfer to taxa belonging to the predicted evolutionary host range of the plasmids was not observed. Overall, the ARG dissemination potential uncovered in WWTP communities calls for a thorough risk assessment of ARG transmission across the wastewater system, before identification of possible mitigation strategies.

The herbicide bentazone is recalcitrant in aquifers and is therefore frequently detected in wells used for drinking water production. However, bentazone degradation has been observed in filter sand from a rapid sand filter at a waterworks with methane-rich groundwater. Here, the association between methane oxidation and removal of bentazone was investigated with a methanotrophic enrichment culture derived from methane-fed column reactors inoculated with that filter sand. Several independent lines of evidence obtained from microcosm experiments with the methanotrophic enrichment culture, tap water and bentazone at concentrations below 2 mg/L showed methanotrophic co-metabolic bentazone transformation: The culture removed 53% of the bentazone in 21 days in presence of 5 mg/L of methane, while only 31% was removed in absence of methane. Addition of acetylene inhibited methane oxidation and stopped bentazone removal. The presence of bentazone partly inhibited methane oxidation since the methane consumption rate was significantly lower at high (1 mg/L) than at low (1 μg/L) bentazone concentrations. The transformation yield of methane relative to bentazone normalized by their concentration ratio ranged from 58 to 158, well within the range for methanotrophic co-metabolic degradation of trace contaminants calculated from the literature, with normalized substrate preferences varying from 3 to 400. High-resolution mass spectrometry revealed formation of the transformation products (TPs) 6-OH, 8-OH, isopropyl-OH and di-OH-bentazone, with higher abundances of all TPs in the presence of methane. Overall, we found a suite of evidence all showing that bentazone was co-metabolically transformed to hydroxy-bentazone by a methanotrophic culture enriched from a rapid sand filter at a waterworks.

The recent discovery of completely nitrifying Nitrospira demands a re-examination of nitrifying environments to evaluate their contribution to nitrogen cycling. To approach this challenge, tools are needed to detect and quantify comammox Nitrospira. We present primers for the simultaneous quantification and diversity assessement of both comammox Nitrospira clades. The primers cover a wide range of comammox diversity, spanning all available high quality sequences. We applied these primers to 12 groundwater-fed rapid sand filters, and found comammox Nitrospira to be abundant in all filters. Clade B comammox comprise the majority (∼75%) of comammox abundance in all filters. Nitrosomonadaceae were present in all filters, although at low abundance (mean = 1.8%). Ordination suggests that temperature impacts the structure of nitrifying communities, and in particular that increasing temperature favours Nitrospira. The nitrogen content of the filter material, sulfate concentration and surface ammonium loading rates shape the structure of the comammox guild in the filters. This work provides an assay for simultaneous detection and diversity assessment of clades A and B comammox Nitrospira, expands our current knowledge of comammox Nitrospira diversity and demonstrates a key role for comammox Nitrospira in nitrification in groundwater-fed biofilters.

Summary Community assembly is a central topic in microbial ecology: how do assembly processes interact and what is the relative contribution of stochasticity and determinism? Here, we exposed replicate flow-through biofilm systems, fed with nitrite-supplemented tap water, to continuous immigration from a source community, present in the tap water, to determine the extent of selection and neutral processes in newly assembled biofilm communities at both the community and the functional guild (of nitrite-oxidizing bacteria, NOB) levels. The community composition of biofilms assembled under low and high nitrite loading was described after 40 days of complete nitrite removal. The total community assembly, as well as the NOB guild assembly were largely governed by a combination of deterministic and stochastic processes. Furthermore, we observed deterministic enrichment of certain types of NOB in the biofilms. Specifically, elevated nitrite loading selected for a single Nitrotoga representative, while lower nitrite conditions selected for a number of Nitrospira. Therefore, even when focusing on ecologically coherent ensembles, assembly is the result of complex stochastic and deterministic processes that can only be interrogated by observing multiple assemblies under controlled conditions. This article is protected by copyright. All rights reserved.

Copper is a cofactor of the ammonia monooxygenase, an essential enzyme for the activity of ammonia oxidizing prokaryotes (AOP). Copper dosing at less than 1 μg/L stimulated ammonium removal in the poorly nitrifying biological filters of three full-scale drinking water treatment plants. Upon copper dosing, the ammonium concentration in the effluent decreased from up to 0.18 to less than 0.01 mg NH

Nitrification was long thought to consist in the stepwise oxidation of ammonia to nitrite and of nitrite to nitrate by ammonia oxidizing and nitrite oxidizing microorganisms, respectively. Recently, single microorganisms capable of complete ammonia to nitrate oxidation (comammox) were identified in theNitrospiragenus. This genus, previously considered to only contain canonical nitrite oxidizers is diverse and has a broad environmental distribution. Yet, a global insight into the abundance, niche preference, and genomic diversity ofNitrospirais missing. Here, we established the largestNitrospiragenome database to date, revealing 68 putative species, most without cultivated representatives. We performed a global survey through read recruitment of metagenomic data from various environments against this database that identified that environmental filtering structures species distribution, without large scale biogeographical signal. The ecological success of comammoxNitrospirais evident as they outnumber and are more diverse than canonicalNitrospirain communities from all environments but wastewater treatment plants. We detect a phylogenetic signal inNitrospiraspecies habitat preference, that is strongest for canonicalNitrospiraspecies. ComammoxNitrospiraeco-evolutionary history is more complex with subclades achieving rapid niche divergence via horizontal transfer of genes, including that encoding the hydroxylamine oxidoreductase, one of the key enzymes involved in nitrification.

The ability to disperse is considered essential for soil bacteria colonization and survival, yet very little is known about the dispersal ability of communities from different heterogeneous soil compartments. Important factors for dispersal are the thickness and connectivity of the liquid film between soil particles. The present results from a fractured clay till depth profile suggest that dispersal ability is common in various soil compartments and that most are dominated by a few dispersing taxa. Importantly, an increase in shared dispersers among the preferential flow paths of the clay till suggests that active dispersal plays a role in the successful colonization of these habitats., This study assessed the dispersal of five bacterial communities from contrasting compartments along a fractured clay till depth profile comprising plow layer soil, preferential flow paths (biopores and the tectonic fractures below), and matrix sediments, down to 350 cm below the surface. A recently developed expansion of the porous surface model (PSM) was used to capture bacterial communities dispersing under controlled hydration conditions on a soil-like surface. All five communities contained bacteria capable of active dispersal under relatively low hydration conditions (−3.1 kPa). Further testing of the plow layer community revealed active dispersal even at matric potentials of −6.3 to −8.4 kPa, previously thought to be too dry for dispersal on the PSM. Using 16S rRNA gene amplicon sequencing, the dispersing communities were found to be less diverse than their corresponding total communities. The dominant dispersers in most compartments belonged to the genus Pseudomonas and, in the plow layer soil, to Rahnella as well. An exception to this was the dispersing community in the matrix at 350 cm below the surface, which was dominated by Pantoea. Hydrologically connected compartments shared proportionally more dispersing than nondispersing amplicon sequence variants (ASVs), suggesting that active dispersal is important for colonizing these compartments. These results highlight the importance of including soil profile heterogeneity when assessing the role of active dispersal and contribute to discerning the importance of active dispersal in the soil environment. IMPORTANCE The ability to disperse is considered essential for soil bacteria colonization and survival, yet very little is known about the dispersal ability of communities from different heterogeneous soil compartments. Important factors for dispersal are the thickness and connectivity of the liquid film between soil particles. The present results from a fractured clay till depth profile suggest that dispersal ability is common in various soil compartments and that most are dominated by a few dispersing taxa. Importantly, an increase in shared dispersers among the preferential flow paths of the clay till suggests that active dispersal plays a role in the successful colonization of these habitats.

Copper is a co-factor of the ammonia monooxygenase, an essential enzyme for the activity of ammonia oxidizing prokaryotes (AOP). Copper dosing at less than 1 µg/L stimulated ammonium removal in the poorly-nitrifying biological filters of three full-scale drinking water treatment plants. Upon copper dosing, the ammonium concentration in the effluent decreased from up to 0.18 to less than 0.01 mg NH4+-N/L. To investigate how copper dosing affected the filter microbial community, we applied amplicon sequencing and qPCR targeting key nitrifying groups, including complete ammonia oxidizing (comammox) Nitrospira. Copper dosing increased the abundance of different nitrifiers. Multiple Nitrosomonas variants (betaproteobacterial ammonia oxidizers), which initially collectively represented 1% or less of the total community, increased almost 10 fold. Comammox Nitrospira were abundant and increased too, but their relative abundance within the AOP decreased because of Nitrosomonas proliferation. No other consistent change in the filter communities was detected, as well as no adverse effect of copper on the filters functionality. Our results show that copper dosing in three independent plants was associated with consistent growth of AOP and that efficient nitrification was achieved through the joint contribution of comammox Nitrospira and an increasing fraction of betaproteobacterial ammonia oxidizers.

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.

In this study, we developed a method that provides profiles of community-level surface dispersal from environmental samples under controlled hydration conditions and enables us to isolate and uncover the diversity of the fastest bacterial dispersers. The method expands on the porous surface model (PSM), previously used to monitor the dispersal of individual bacterial strains in liquid films at the surface of a porous ceramic disc. The novel procedure targets complex communities and captures the dispersed bacteria on a solid medium for growth and detection. The method was first validated by distinguishing motile Pseudomonas putida and Flavobacterium johnsoniae strains from their nonmotile mutants. Applying the method to soil and lake water bacterial communities showed that community-scale dispersal declined as conditions became drier. However, for both communities, dispersal was detected even under low-hydration conditions (matric potential, −3.1 kPa) previously proven too dry for P. putida strain KT2440 motility. We were then able to specifically recover and characterize the fastest dispersers from the inoculated communities. For both soil and lake samples, 16S rRNA gene amplicon sequencing revealed that the fastest dispersers were substantially less diverse than the total communities. The dispersing fraction of the soil microbial community was dominated by Pseudomonas species cells, which increased in abundance under low-hydration conditions, while the dispersing fraction of the lake community was dominated by Aeromonas species cells and, under wet conditions (−0.5 kPa), also by Exiguobacterium species cells. The results gained in this study bring us a step closer to assessing the dispersal ability within complex communities under environmentally relevant conditions. IMPORTANCE Dispersal is a key process of bacterial community assembly, and yet, very few attempts have been made to assess bacterial dispersal at the community level, as the focus has previously been on pure-culture studies. A crucial factor for dispersal in habitats where hydration conditions vary, such as soils, is the thickness of the liquid films surrounding solid surfaces, but little is known about how the ability to disperse in such films varies within bacterial communities. Therefore, we developed a method to profile community dispersal and identify fast dispersers on a rough surface resembling soil surfaces. Our results suggest that within the motile fraction of a bacterial community, only a minority of the bacterial types are able to disperse in the thinnest liquid films. During dry periods, these efficient dispersers can gain a significant fitness advantage through their ability to colonize new habitats ahead of the rest of the community.

Universal (i.e., targeting most bacteria/prokaryotes) 16S rRNA gene based amplicon sequencing is widely used for assessing microbial communities due to its low cost, time efficiency, and ability to provide a full overview of the community. However, it is currently unclear if it can yield reliable information on specific microbial guilds, as obtained by using primer sets targeting functional genes or specific16S rRNA gene sequences. Here, we compared the relative abundance, diversity, richness, and composition of selected guilds (nitrifiers), obtained from universal 16S rRNA gene based amplicon sequencing and from guild targeted approaches. The universal amplicon sequencing provided 1) accurate estimates of nitrifier composition, 2) clustering of the samples based on these compositions consistent with sample origin, 3) estimates of the relative abundance of the guilds correlated with those obtained from the targeted approaches and within ~1.2 orders of magnitude of them, but with measurable bias that should be considered when comparing estimates from both approaches. In contrast, the diversity and richness estimations using the universal 16S rRNA based amplicon sequencing were likely limited by the sequencing depth; therefore, we suggest preferring targeted approaches for assessing nitrifiers diversity and richness or using sequencing depth larger than those currently typically practiced. Overvall, we conclude that universal amplicon sequencing provides, in a single analysis, useful information on the abundance and composition of diverse guilds in complex environmental communities.

The description of comammoxNitrospiraspp., performing complete ammonium-to-nitrate oxidation, and their co-occurrence with canonical betaproteobacterial ammonium oxidizing bacteria (β-AOB) in the environment, call into question the metabolic potential of comammoxNitrospiraand the evolutionary history of their ammonium oxidation pathway. We report four new comammoxNitrospiragenomes, constituting two novel species, and the first comparative genomic analysis on comammoxNitrospira.ComammoxNitrospirahas lost the potential to use external nitrite as energy and nitrogen source: compared to strictly nitrite oxidizingNitrospira; they lack genes for assimilative nitrite reduction and reverse electron transport from nitrite. By contrast, compared to otherNitrospira, their ammonium oxidizer physiology is exemplified by genes for ammonium and urea transporters and copper homeostasis and the lack of cyanate hydratase genes. Two comammox clades are different in their ammonium uptake systems. Contrary to β-AOB, comammoxNitrospiragenomes have single copies of the two central ammonium oxidation pathway genes, lack genes involved in nitric oxide reduction, and encode genes that would allow efficient growth at low oxygen concentrations. Hence, comammoxNitrospiraseems attuned to oligotrophy and hypoxia compared to β-AOB.β-AOBs are the clear origin of the ammonium oxidation pathway in comammoxNitrospira: reconciliation analysis indicates two separate earlyamoAgene transfer events from β-AOB to an ancestor of comammoxNitrospira, followed by clade specific losses. ForhaoA, one early transfer from β-AOB to comammoxNitrospirais predicted – followed by intra-clade transfers. We postulate that the absence of comammox genes in mostNitrospiragenomes is the result of subsequent loss.SignificanceThe recent discovery of comammox bacteria - members of theNitrospiragenus able to fully oxidize ammonia to nitrate - upset the long-held conviction that nitrification is a two-step process. It also opened key questions on the ecological and evolutionary relations of these bacteria with other nitrifying prokaryotes. Here, we report the first comparative genomic analysis of comammoxNitrospiraand related nitrifiers. Ammonium oxidation genes in comammoxNitrospirahad a surprisingly complex evolution, originating from ancient transfer from the phylogenetically distantly related ammonia-oxidizing betaproteobacteria, followed by within-lineage transfers and losses. The resulting comammox genomes are uniquely adapted to ammonia oxidation in nutrient-limited and low-oxygen environments and appear to have lost the genetic potential to grow by nitrite oxidation alone.

Despite recent efforts in identifying the determinants of invasion in microbial communities, experimental observations across different ecosystems are inconclusive. While relationships between resident community diversity and invasion success are often noted, community diversity says little about community assembly processes. Community assembly processes may provide a more inclusive framework to explain—and potentially prevent or facilitate—invasion. Here we let replicate nitrite-oxidizing bacterial guilds assemble under different conditions from a natural source community and study their compositional patterns to infer the relative importance of the assembly processes. Then, an invader strain from that same guild was introduced at one of three propagule pressures. We found no significant correlation between community diversity and invasion success. Instead, we observed that the effect of selection on invasion success was surpassed by the effect of drift, as inferred from the substantial influence of propagule pressure on invasion success. This dominance of drift can probably be generalized to other invasion cases with high phylogenetic similarity between invader and resident community members. In these situations, our results suggest that attempting to modulate the invasibility of a community by altering its diversity is futile because stochastic processes determine the invasion outcome. Increasing or reducing propagule pressure is then deemed the most efficient avenue to enhance or limit invasion success.

Steep physiochemical gradients and diffusive limitation associated with microscale features such as cracks and pores make soil and sediments remarkably heterogeneous environments, which is reflected on many environmentally important processes. If we are to understand and attempt to control the ecology of the microorganisms which inhabit these environments we must not only characterize their inhabitants, but also the complex biogeochemical landscape they live in. This includes local concentrations of electron acceptors and donors, microbial metabolites and key physical and chemical parameters such as pH and soil structure. To this end, an array of techniques for collecting data at the microscale has been developed, deployed and refined, ranging from microsensor probes to planar sensors. This review provides a general reference for and a critical comparison of microscale techniques available to the fields of soil and sediment microbial ecology. Techniques are evaluated based on their ability to provide spatially resolved data at the microscale, with focus on performance characteristics, potential for repeated measurements, degree of physical disruption they create, and accessibility. Microscale studies have given us many insights, but we outline further progress needed to make the microscale toolkit more accessible and to extend the range of analytes that can be measured simultaneously, so that we may expand our knowledge of the complex environmental microscale heterogeneity and its impact on soil and sediment ecology and functioning.

The objective of this work is to identify relevant settling velocity and rheology model parameters and to assess the underlying filamentous microbial community characteristics that can influence the solids mixing and transport in secondary settling tanks. Parameter values for hindered, transient and compression settling velocity functions were estimated by carrying out biweekly batch settling tests using a novel column setup through a four-month long measurement campaign. To estimate viscosity model parameters, rheological experiments were carried out on the same sludge sample using a rotational viscometer. Quantitative fluorescence in-situ hybridisation (qFISH) analysis, targeting Microthrix parvicella and phylum Chloroflexi, was used. This study finds that M. parvicella - predominantly residing inside the microbial flocs in our samples - can significantly influence secondary settling through altering the hindered settling velocity and yield stress parameter. Strikingly, this is not the case for Chloroflexi, occurring in more than double the abundance of M. parvicella, and forming filaments primarily protruding from the flocs. The transient and compression settling parameters show a comparably high variability, and no significant association with filamentous abundance. A two-dimensional, axi-symmetrical computational fluid dynamics (CFD) model was used to assess calibration scenarios to model filamentous bulking. Our results suggest that model predictions can significantly benefit from explicitly accounting for filamentous bulking by calibrating the hindered settling velocity function. Furthermore, accounting for the transient and compression settling velocity in the computational domain is crucial to improve model accuracy when modelling filamentous bulking. However, the case-specific calibration of transient and compression settling parameters as well as yield stress is not necessary, and an average parameter set - obtained under bulking and good settling conditions - can be used.

The environmental stimulants and inhibitors of conjugal plasmid transfer in microbial communities are poorly understood. Specifically, it is not known whether exposure to stressors may cause a community to alter its plasmid uptake ability. We assessed whether metals (Cu, Cd, Ni, Zn) and one metalloid (As), at concentrations causing partial growth inhibition, modulate community permissiveness (that is, uptake ability) against a broad-host-range IncP-type plasmid (pKJK5). Cells were extracted from an agricultural soil as recipient community and a cultivation-minimal filter mating assay was conducted with an exogenous E. coli donor strain. The donor hosted a gfp-tagged pKJK5 derivative from which conjugation events could be microscopically quantified and transconjugants isolated and phylogenetically described at high resolution via FACS and 16S rRNA amplicon sequencing. Metal stress consistently decreased plasmid transfer frequencies to the community, while the transconjugal pool richness remained unaffected with OTUs belonging to 12 bacterial phyla. The taxonomic composition of the transconjugal pools was distinct from their respective recipient communities and clustered dependent on the stress type and dose. However, for certain OTUs, stress increased or decreased permissiveness by more than 1000-fold and this response was typically correlated across different metals and doses. The response to some stresses was, in addition, phylogenetically conserved. This is the first demonstration that community permissiveness is sensitive to metal(loid) stress in a manner that is both partially consistent across stressors and phylogenetically conserved.The ISME Journal advance online publication, 2 August 2016; doi:10.1038/ismej.2016.98.

Until recently, nitrification was thought to be a strict two-step process where ammonia was first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite oxidizing bacteria (NOB). Recent studies in NOB metabolism, however, have revealed that certain Nitrospira are capable of performing both steps, resulting in complete ammonia oxidation (comammox) by single microorganisms. These comammox Nitrospira have been detected in drinking water (Pinto et al., 2015; Palomo et al., 2016) and aquaculture systems (van Kessel et al., 2015), as well as deep oil exploration wells (Daims et al., 2015). The discovery of comammox Nitrospira has significantly changed our understanding of biogeochemical nitrogen cycle. The goal of this experiment was to determine the extent of competition between comammox Nitrospira and canonical Nitrospira in ammonium scarce environment, with nitrite as the main energy source. Community assembly was monitored on well-established biofilms formed on the grains of rapid sand filter (RSF) for drinking water production. RSF sand was placed in laboratory scale column bioreactors and subjected to continuous feeding of tap water spiked with NO2- (1 mg-N/L) for 250 days. The biofilms were then characterized by Illumina MiSeq platform, targeting the 16S rRNA gene. The relative abundance of a putative comammox clade B Nitrospira sequence variant (with 100% 16S rRNA gene similarity to comammox CG24_A assembled genome) identified in the initial RSF sand (Palomo et al., unpublished) at a relative abundance of 12.4±1.1%, was not detected in 4 out of 6 replicates after 250 days. Similar trend was observed for other putative comammox clade B Nitrospira sequence variants. In contrast, we observed significant increase (padj

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.

Pseudomonas putida is a versatile bacterial species adapted to soil and its fluctuations. Like many other species living in soil, P. putida often faces water limitation. Alginate, an exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect of water limitation. In addition to alginate, P. putida is capable of producing cellulose (bcs), putida exopolysaccharide a (pea), and putida exopolysaccharide b (peb). However, unlike alginate, not much is known about their roles under water limitation. Hence, in this study we examined the role of different EPS components under mild water limitation. To create environmentally realistic water limited conditions as observed in soil, we used the Pressurized Porous Surface Model. Our main hypothesis was that under water limitation and in the absence of alginate other exopolysaccharides would be more active to maintain homeostasis. To test our hypothesis, we investigated colony morphologies and whole genome transcriptomes of P. putida KT2440 wild type and its mutants deficient in synthesis of either alginate or all known EPS. Overall our results support that alginate is an important exopolysaccharide under water limitation and in the absence of alginate other tolerance mechanisms are activated.