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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.

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.

Membrane-aerated biofilm reactors (MABRs) are a novel technology based on the growth of biofilms on oxygen-permeable membranes. Hereby, MABRs combine all the advantages of biofilm growth with a more flexible and efficient control of the oxygen load. In the present work, MABR flow cells were operated to achieve full nitrification. MABR biofilms had a significantly different structure than co-diffusion biofilms reported in the literature. Different levels of shear stress and oxygen loading during MABR operation also affected the biofilm parameters. Furthermore, reactor operation at higher oxygen loads resulted in an increased biofilm cohesiveness, which depended on the EPS mass in the biofilms and the type of stress applied (more cohesive against normal than shear stresses). The EPS in the strongest biofilms had a higher content of proteins and a lower level of carbohydrates. Staining analyses revealed that the outermost EPS in the stronger biofilm regions was of hydrophilic nature and distributed around dense microbial aggregates, whereas it was homogeneously distributed in the weaker strata. Overall, the obtained results provide input parameters to future modelling efforts and operating conditions to support more robust autotrophic N conversions in MABRs.

The potential for pesticide degradation varies greatly at the centimeter-scale in agricultural soil. Three dimensional numerical simulations were conducted to evaluate how such small-scale spatial heterogeneity may affect the leaching of the biodegradable pesticide 2-methyl-4-chlorophenoxyacetic acid (MCPA) in the upper meter of a variably-saturated, loamy soil profile. To incorporate realistic spatial variation in degradation potential, we used data from a site where 420 mineralization curves over 5 depths have been measured. Monod kinetics was fitted to the individual curves to derive initial degrader biomass values, which were incorporated in a reactive transport model to simulate heterogeneous biodegradation. Six scenarios were set up using COMSOL Multiphysics to evaluate the difference between models having different degrader biomass distributions (homogeneous, heterogeneous, or no biomass) and either matrix flow or preferential flow through a soil matrix with a wormhole. MCPA leached, within 250 days, below 1m only when degrader biomass was absent and preferential flow occurred. Both biodegradation in the plow layer and the microbially active lining of the wormhole contributed to reducing MCPA-leaching below 1m. The spatial distribution of initial degrader biomass within each soil matrix layer, however, had little effect on the overall MCPA-leaching.

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.

Seasonal changes in arsenic and iron accumulation rates were examined in the sediments of a brook that receives groundwater discharges of arsenic and reduced iron. Clean glass bead columns were deployed in sediments for known periods over the annual hydrologic cycle to monitor changes in arsenic and iron concentrations in bead coatings. The highest accumulation rates occurred during the dry summer period (July-October) when groundwater discharges were likely greatest at the sample locations. The intermediate flow period (October-March), with higher surface water levels, was associated with losses of arsenic and iron from bead column coatings at depths below 2-6 cm. Batch incubations indicated iron releases from solids to be induced by biological reduction of iron (oxy)hydroxide solids. Congruent arsenic releases during incubation were limited by the high arsenic sorption capacity (0.536 mg(As)/mg(Fe)) of unreacted iron oxide solids. The flooded spring (March-June) with high surface water flows showed the lowest arsenic and iron accumulation rates in the sediments. Comparisons of accumulation rates across a shoreline transect were consistent with greater rates at regions exposed above surface water levels for longer times and greater losses at locations submerged below surface water. Iron (oxy)hydroxide solids in the shallowest sediments likely serve as a passive barrier to sorb arsenic released to pore water at depth by biological iron reduction.

A multispecies one-dimensional biofilm model considering nitric oxide (NO) and nitrous oxide (N2O) productions for membrane aerated biofilm reactor (MABR) that remove nitrogen autotrophically through aerobic ammonia oxidation followed by Anammox is used to study the role of Anammox activity on the total nitrogen (TN) removal and the productions of NO and N2O. The model is applied to evaluate how periodic aeration as a control parameter reduces NO and N2O production but maintains high TN removal in MABR. The simulation results show over 3.5% of the removed TN could be attributed to NO and N2O production in MABR under the operational conditions optimal for TN removal (72%). An analysis of factors governing the Anammox activity in MABR shows that enhancing Anammox activity not only helps to achieve a high level of nitrogen removal but also reduces NO and N2O productions. Comparison of aeration strategies (periodic aeration vs. continuous aeration) reveals that periodic aeration can reduce NO and N2O production while maintaining a high level of nitrogen removal through promoting Anammox growth. Application of periodic aerations with different cycle frequencies to the MABR indicates that an increase in the cycle frequency of the periodic aeration can further maximize TN removal and minimize the NO and N2O production in membrane aerated biofilm. The information of this paper will be useful for understanding the indirect role of Anammox on NO and N2O productions and for optimizing the design and operation of MABR systems. (C) 2013 Elsevier B.V. All rights reserved

The remarkable oxygen transfer efficiencies attainable in membrane-aerated biofilm reactors (MABRs) are expected to favor their prompt industrial implementation. However, tests in clean water, currently used for the estimation of their oxygen transfer potential, lead to wrong estimates once biofilm is present, significantly complicating reactor modeling and control. This study shows for the first time the factors affecting oxygen mass transfer across membranes during clean water tests and reactor operation via undisturbed microelectrode inspection and bulk measurements. The mass transfer resistance of the liquid boundary layer developed at the membrane–liquid interface during clean water tests accounted for two thirds of the total mass transfer resistance, suggesting a strong underestimation of the oxygen transfer rates when it is absent (e.g. after biofilm growth). Reactor operation to attain partial nitritation showed that predicted oxygen transfer rates are enhanced up to six times with biofilm activity. The higher availability of ammonia at the biofilm base drives this process. Such behavior can be captured with the addition of two terms (depending on system characteristics and reactor loading) to existing model structures. Overall, we provide tools to better estimate, model, and optimize oxygen transfer supporting a more energy-efficient approach to MABR operation.

Membrane-aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriouslyhampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long-term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real-time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10-fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diversethan NOB, and dominated by the r-strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor andcontrol the microbial communities that support robust, sustainable and efficient nitrogen removal.

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.

This work describes the successful coupling of partial nitrification (nitritation) and anaerobic ammonium oxidation in a membrane-aerated biofilm reactor (MABR) with continuous aeration. Controlling the relative surface loadings of oxygen versus ammonium prevented complete nitrite oxidation and allowed anaerobic ammonium-oxidizing bacteria (AnaerAOB) to develop and be retained for >250 days. Daily autotrophic nitrogen removal of 1.7 g N/m2 (75% of influent N load) was achieved at an oxygen/nitrogen surface loading ratio of 2.2, with up to 85% of the influent N proceeding through AnaerAOB. During early nitritation, nitrogen oxide (NO(g), NO2(g), and N2O(g)) emissions comprised up to 10% of the removed influent nitrogen, but emissions disappeared after proliferation of AnaerAOB. Microbial communities were radially stratified, with aerobic ammonium-oxidizing bacteria (AerAOB) colonizing nearest to and AnaerAOB furthest from the membrane. Despite the presence of nitrite-oxidizing bacteria, this work ...

The effect of biokinetics on nitritation was investigated in two biofilm geometries, the Membrane Aerated Biofilm Reactor (MABR) and a conventional biofilm system. A 1D biofilm model was used and evaluated by global sensitivity analysis using the variance based Sobol method. The main focus was on the influence of key biokinetic parameters (maximum specific growth rates, oxygen and nitrogen affinity constants of AOB (ammonium oxidizing bacteria) and NOB (nitrite oxidizing bacteria)) and their ratios on nitritation efficiency in these geometries. This exhaustive simulation study revealed that nitritation strongly depends on the chosen kinetic parameters of AOB and NOB. The maximum specific growth rates (μmax,AOB and μmax,NOB) had the strongest impact on nitritation efficiency (NE). In comparison, the counter-diffusion geometry yielded more parameter combinations (27.5%) that resulted in high NE than the co-diffusion geometry (7.9%). The oxygen concentrations at the relevant biofilm interfaces (membrane/biofilm for counter-diffusion or bulk/biofilm for co-diffusion) were not predictive of NE. However, the maximum allowable oxygen concentration to maintain higher NE was higher for the counter-diffusion geometry.

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.

Allylthiourea (ATU) and chlorate (ClO 3 −) are often used to selectively inhibit nitritation and nitratation. In this work we identified challenges with use of these compounds in inhibitory assays with filter material from a biological rapid sand filter for groundwater treatment. Inhibition was investigated in continuous-flow lab-scale columns, packed with filter material from a full-scale filter and supplied with NH 4 + or NO 2 −. ATU concentrations of 0.1–0.5 mM interfered with the indophenol blue method for NH 4 + quantification leading to underestimation of the measured NH 4 + concentration. Interference was stronger at higher ATU levels and resulted in no NH 4 + detection at 0.5 mM ATU. ClO 3 − at typical concentrations for inhibition assays (1–10 mM) inhibited nitratation by less than 6%, while nitritation was instead inhibited by 91% when NH 4 + was supplied. On the other hand, nitratation was inhibited by 67–71% at 10–20 mM ClO 3 − when NO 2 − was supplied, suggesting significant nitratation inhibition at higher NO 2 − concentrations. No chlorite (ClO 2 −) was detected in the effluent, and thus we could not confirm that nitritation inhibition was caused by ClO 3 − reduction to ClO 2 −. In conclusion, ATU and ClO 3 − should be used with caution in inhibition assays, because analytical interference and poor selectivity for the targeted process may affect the experimental outcome and compromise result interpretation.

There is a growing interest in controlling—promoting or avoiding—the invasion of microbial communities by new community members. Resource availability and community structure have been reported as determinants of invasion success. However, most invasion studies do not adhere to a coherent and consistent terminology nor always include rigorous interpretations of the processes behind invasion. Therefore, we suggest that a consistent set of definitions and a rigorous conceptual framework are needed. We define invasion in a microbial community as the establishment of an alien microbial type in a resident community and argue how simple criteria to define aliens, residents, and alien establishment can be applied for a wide variety of communities. In addition, we suggest an adoption of the community ecology framework advanced by Vellend (2010) to clarify potential determinants of invasion. This framework identifies four fundamental processes that control community dynamics: dispersal, selection, drift and diversification. While selection has received ample attention in microbial community invasion research, the three other processes are often overlooked. Here, we elaborate on the relevance of all four processes and conclude that invasion experiments should be designed to elucidate the role of dispersal, drift and diversification, in order to obtain a complete picture of invasion as a community process.

Nitrous oxide (N

Third generation biofuels, e.g. biofuels production from algal biomass, have gained attention due to increased interest on global renewable energy. However, crop-based biofuels compete with food production and should be avoided. Microalgal cultivation for biofuel production offers an alternative to crops and can become economically viable when combined with the use of used water resources. Besides nutrients and water, harvesting microalgal biomass represents one of the major costs related to biofuel production and thus efficient and cheap solutions are needed. In bacterial-algal systems, there is the potential to produce energy by co-digesting the two types of biomass. We present an innovative approach to recover microalgal biomass via a two-step flocculation using bacterial biomass after the destabilisation of microalgae with conventional cationic polymer. A short solids retention time (SRT) enhanced biological phosphorus removal (EBPR) system was combined with microalgal cultivation. Two different bacterial biomass removal strategies were assessed whereby bacterial biomass was collected from the solid-liquid separation after the anaerobic phase and after the aerobic phase. Microalgal recovery was tested by jar tests where three different chemical coagulants in coagulation-flocculation tests (AlCl3, PDADMAC and Greenfloc 120) were assessed. Furthermore, jar tests were conducted to assess the microalgal biomass recovery by a two-step flocculation method, involving chemical coagulants in the first step and bacterial biomass used in the second step to enhance the flocculation. Up to 97% of the microalgal biomass was recovered using 16 mg polymer/g algae and 0.1 g algae/g bacterial biomass. Moreover, the energy recovery by the short-SRT EBPR system combined with microalgal cultivation was assessed via biomethane potential tests. Up to 560 ± 24 mL CH4/gVS methane yield was obtained by co-digesting bacterial biomass collected after the anaerobic phase and microalgal biomass. The energy recovery in terms of methane production obtained in the short-SRT EBPR system is about 40% of the influent chemical energy.

Four main processes are considered to drive microbial community assembly: selection, drift, dispersal and speciation. These processes occur simultaneously, but the extent to which each process contributes to community assembly is unclear in natural communities. We exposed a high-throughput flow-through biofilm system to continuous immigration from a tap water metacommunity while applying different nitrite surface loading rates. After 63 days of operation, we extracted biofilms and analyzed the community composition via Illumina MiSeq targeting the 16S rRNA gene. Previous studies have shown that Nitrospira is the dominant nitrite oxidizing genus in low nitrite environments. Hence, we postulated that by elevating the nitrite surface loading we would select for NOB with lower nitrite affinity than Nitrospira. We observed different dominant NOB species under different loading rates. While in the metacommunity, Nitrotoga and Nitrospira were found at near equal abundances, in the biofilm community, elevated nitrite loading strongly selected for Nitrotoga over Nitrospira. The biofilms were also significantly different in their alpha-diversity (p

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.

Process models describing nitrous oxide (N2O) production during biological nitrogen removal allow for the development of mitigation strategies of this potent greenhouse gas. N2O is an intermediate of nitrogen removal, hence its prediction is negatively affected by the uncertainty associated to its substrates. Improving experimental designs for model calibration reduces prediction uncertainties. Moreover, the individual analysis of autotrophic and heterotrophic contribution to the total NO and N2O pool was assessed for already proposed model structures under different experimental scenarios. The results show the need for information-rich experiemental designs to assess the predicting capabilities of N2O models. This work represents a step further in understanding the N2O production and emissions associated to conventional wastewater treatment. Moreovere, it will facilitate the development of strategies to minimize the carbon footprint of wastewater treatment plants.

Rapid gravity sand filtration is a drinking water production technology widely used around the world. Microbially catalyzed processes dominate the oxidative transformation of ammonia, reduced manganese and iron, methane and hydrogen sulfide, which may all be present at millimolar concentrations when groundwater is the source water. In this study, six metagenomes from various locations within a groundwater-fed rapid sand filter (RSF) were analyzed. The community gene catalog contained most genes of the nitrogen cycle, with particular abundance in genes of the nitrification pathway. Genes involved in different carbon fixation pathways were also abundant, with the reverse tricarboxylic acid cycle pathway most abundant, consistent with an observed Nitrospira dominance. From the metagenomic data set, 14 near-complete genomes were reconstructed and functionally characterized. On the basis of their genetic content, a metabolic and geochemical model was proposed. The organisms represented by draft genomes had the capability to oxidize ammonium, nitrite, hydrogen sulfide, methane, potentially iron and manganese as well as to assimilate organic compounds. A composite Nitrospira genome was recovered, and amo-containing Nitrospira genome contigs were identified. This finding, together with the high Nitrospira abundance, and the abundance of atypical amo and hao genes, suggests the potential for complete ammonium oxidation by Nitrospira, and a major role of Nitrospira in the investigated RSFs and potentially other nitrifying environments.

In biofilm systems for wastewater treatment (e.g., moving bed biofilms reactors-MBBRs) biofilm thickness is typically not under direct control. Nevertheless, biofilm thickness is likely to have a profound effect on the microbial diversity and activity, as a result of diffusion limitation and thus substrate penetration in the biofilm. In this study, we investigated the impact of biofilm thickness on nitrification and on the removal of more than 20 organic micropollutants in laboratory-scale nitrifying MBBRs. We used novel carriers (Z-carriers, AnoxKaldnes) that allowed controlling biofilm thickness at 50, 200, 300, 400, and 500 μm. The impact of biofilm thickness on microbial community was assessed via 16S rRNA gene amplicon sequencing and ammonia monooxygenase (amoA) abundance quantification through quantitative PCR (qPCR). Results from batch experiments and microbial analysis showed that (i) the thickest biofilm (500 μm) presented the highest specific biotransformation rate constants (kbio, L g(-1) d(-1)) for 14 out of 22 micropollutants; (ii) biofilm thickness positively associated with biodiversity, which was suggested as the main factor for the observed enhancement of kbio; (iii) the thinnest biofilm (50 μm) exhibited the highest nitrification rate (gN d(-1) g(-1)), amoA gene abundance and kbio values for some of the most recalcitrant micropollutants (i.e., diclofenac and targeted sulfonamides). Although thin biofilms favored nitrification activity and the removal of some micropollutants, treatment systems based on thicker biofilms should be considered to enhance the elimination of a broad spectrum of micropollutants.

The new paradigm for used water treatment suggests the use of short solid retention times (SRT) to minimize organic substrate mineralization and to maximize resource recovery. However, little is known about the microbes and the underlying biogeochemical mechanisms driving these short-SRT systems. In this paper, we report the start-up and operation of a short-SRT enhanced biological phosphorus removal (EBPR) system operated as a sequencing batch reactor (SBR) fed with preclarified municipal wastewater, which is supplemented with propionate. The microbial community was analysed via 16S rRNA amplicon sequencing. During start-up (SRT = 8 d), the EBPR was removing up to 99% of the influent phosphate and completely oxidized the incoming ammonia. Furthermore, the sludge showed excellent settling properties. However, once the SRT was shifted to 3.5 days nitrification was inhibited and bacteria of the Thiothrix taxon proliferated in the reactor, thereby leading to filamentous bulking (sludge volume index up to SVI = 1100 mL/g). Phosphorus removal deteriorated during this period, likely due to the out-competition of polyphosphate accumulating organisms (PAO) by sulphate reducing bacteria (SRB). Subsequently, SRB activity was suppressed by reducing the anaerobic SRT from 1.2 day to 0.68 day, with a consequent rapid SVI decrease to ∼200 ml/g. The short-SRT EBPR effectively removed phosphate and nitrification was mitigated at SRT = 3 days and oxygen levels ranging from 2 to 3 mg/L.

Ammonia gas is conventionally treated in nitrifying biofilters; however, addition of organic carbon to perform post-denitrification is required to obtain total nitrogen removal. Oxygen-limited autotrophic nitrification/denitrification (OLAND), applied in full-scale for wastewater treatment, can offer a cost-effective alternative for gas treatment. In this study, the OLAND application thus was broadened toward ammonia loaded gaseous streams. A down flow, oxygen-saturated biofilter (height of 1.5 m; diameter of 0.11 m) was fed with an ammonia gas stream (248 ± 10 ppmv) at a loading rate of 0.86 ± 0.04 kg N m(-3) biofilter d(-1) and an empty bed residence time of 14 s. After 45 days of operation a stable nitrogen removal rate of 0.67 ± 0.06 kg N m(-3) biofilter d(-1), an ammonia removal efficiency of 99%, a removal of 75-80% of the total nitrogen, and negligible NO/N(2)O productions were obtained at water flow rates of 1.3 ± 0.4 m(3) m(-2) biofilter section d(-1). Profile measurements revealed that 91% of the total nitrogen activity was taking place in the top 36% of the filter. This study demonstrated for the first time highly effective and sustainable autotrophic ammonia removal in a gas biofilter and therefore shows the appealing potential of the OLAND process to treat ammonia containing gaseous streams.

Water deprivation can be a major stressor to microbial life in surface and subsurface soil. In unsaturated soils, the matric potential (Ψ m ) is often the main component of the water potential, which measures the thermodynamic availability of water. A low matric potential usually translates into water forming thin liquid films in the soil pores. Little is known of how bacteria respond to such conditions, where, in addition to facing water deprivation that might impair their metabolism, they have to adapt their dispersal strategy as swimming motility may be compromised. Using the pressurized porous surface model (PPSM), which allows creation of thin liquid films by controlling Ψ m , we examined the transcriptome dynamics of Pseudomonas putida KT2440. We identified the differentially expressed genes in cells exposed to a mild matric stress (−0.4 MPa) for 4, 24, or 72 h. The major response was detected at 4 h before gradually disappearing. Upregulation of alginate genes was notable in this early response. Flagellar genes were not downregulated, and the microarray data even suggested increasing expression as the stress prolonged. Moreover, we tested the effect of polyethylene glycol 8000 (PEG 8000), a nonpermeating solute often used to simulate Ψ m , on the gene expression profile and detected a different profile than that observed by directly imposing Ψ m . This study is the first transcriptome profiling of KT2440 under directly controlled Ψ m and also the first to show the difference in gene expression profiles between a PEG 8000-simulated and a directly controlled Ψ m .

Summary Plasmid invasion in biofilms is often surprisingly limited in spite of the close contact of cells in a biofilm. We hypothesized that this poor plasmid spread into deeper biofilm layers is caused by a dependence of conjugation on the growth rate (rela- tive to the maximum growth rate) of the donor. By extending an individual-based model of microbial growth and interactions to include the dynamics of plasmid carriage and transfer by individual cells, we were able to conduct in silico tests of this and other hypotheses on the dynamics of conjugal plasmid transfer in biofilms. For a generic model plasmid, we find that invasion of a resident biofilm is indeed limited when plasmid transfer depends on growth, but not so in the absence of growth dependence. Using sensitivity analysis we also find that para- meters related to timing (i.e. a lag before the transconjugant can transfer, transfer proficiency and scan speed) and spatial reach (EPS yield, conjugal pilus length) are more important for successful plasmid invasion than the recipients' growth rate or the probability of segregational loss. While this study identifies one factor that can limit plasmid invasion in biofilms, the new individual-based frame- work introduced in this work is a powerful tool that enables one to test additional hypotheses on the spread and role of plasmids in microbial biofilms.

Nitrous oxide (N2O) can be formed during biological nitrogen (N) removal processes. In this work, a mathematical model is developed that describes N2O production and consumption during activated sludge nitrification and denitrification. The well-known ASM process models are extended to capture N2O dynamics during both nitrification and denitrification in biological N removal. Six additional processes and three additional reactants, all involved in known biochemical reactions, have been added. The validity and applicability of the model is demonstrated by comparing simulations with experimental data on N2O production from four different mixed culture nitrification and denitrification reactor study reports. Modeling results confirm that hydroxylamine oxidation by ammonium oxidizers (AOB) occurs 10 times slower when NO2– participates as final electron acceptor compared to the oxic pathway. Among the four denitrification steps, the last one (N2O reduction to N2) seems to be inhibited first when O2 is present....

Knowledge of the bacterial community structure in sediments is essential to better design restoration strategies for eutrophied lakes. In this regard, the aim of this study was to quantify the abundance and activity of bacteria involved in nutrient and iron cycling in sediments from four Azorean lakes with distinct trophic states (Verde, Azul, Furnas and Fogo). Inferred from quantitative PCR, bacteria performing anaerobic ammonia oxidation were the most abundant in the eutrophic lakes Verde, Azul and Furnas (4.5–16.6%), followed by nitrifying bacteria (0.8–13.0%), denitrifying bacteria (DNB) (0.5–6.8%), iron-reducing bacteria (0.2–1.4%) and phosphorus-accumulating organisms (

Bacterial associations with plant roots are thought to contribute to the success of phytoremediation. We tested the effect of addition of a polycyclic aromatic hydrocarbon contaminated soil on the structure of the rhizosphere microbial communities of wheat (Triticum aestivum), lettuce (Lactuca sativa var. Tango), zucchini (Cucurbita pepo spp. pepo var. Black Beauty), and pumpkin (C. pepo spp. pepo var. Howden) 16S rDNA terminal restriction fragment length polymorphism (T-RFLP) profiles of rhizosphere microbial communities from different soil/plant combinations were compared with a pairwise Pearson correlation coefficient. Rhizosphere microbial communities of zucchini and pumpkin grown in the media amended with highest degree of contaminated soil clustered separately, whereas communities of these plants grown in unamended or amended with lower concentrations of contaminated soil, grouped in a second cluster. Lettuce communities grouped similarly to cucurbits communities, whereas wheat communities did not display an obvious clustering. The variability of 16S rDNA T-RFLP profiles among the different plant/soil treatments were mostly due to the difference in relative abundance rather than presence/absence of T-RFLP fragments. Our results suggest that in highly contaminated soils, the rhizosphere microbial community structure is governed more by the degree of contamination rather than the plant host type.

Summary Individual-based modelling of biofilms accounts for the fact that individual organisms of the same species may well be in a different physiological state as a result of environmental gradients, lag times in responding to change, or noise in gene expression, which we have become increasingly aware of with the advent of single-cell microbiology. But progress in developing and using individual-based modelling has been hampered by different groups writing their own code and the lack of an available standard model. We therefore set out to merge most features of previous models and incorporate various improvements in order to provide a common basis for further developments. Four improvements stand out: the biofilm pressure field allows for shrinking or consolidating biofilms; the continuous-in-time extracellular polymeric substances excretion leads to more realistic fluid behaviour of the extracellular matrix, avoiding artefacts; the stochastic chemostat mode allows comparison of spatially uniform and heterogeneous systems; and the separation of growth kinetics from the individual cell allows condition-dependent switching of metabolism. As an illustration of the model's use, we used the latter feature to study how environmentally fluctuating oxygen availability affects the diversity and composition of a community of denitrifying bacteria that induce the denitrification pathway under anoxic or low oxygen conditions. We tested the hypothesis that the existence of these diverse strategies of denitrification can be explained solely by assuming that faster response incurs higher costs. We found that if the ability to switch metabolic pathways quickly incurs no costs the fastest responder is always the best. However, if there is a trade-off where faster switching incurs higher costs, then there is a strategy with optimal response time for any frequency of environmental fluctuations, suggesting that different types of denitrifying strategies win in different environments. In a single environment, biodiversity of denitrifiers is higher in biofilms than chemostats, higher with than without costs and higher at intermediate frequency of change. The highly modular nature of the new computational model made this case study straightforward to implement, and reflects the sort of novel studies that can easily be executed with the new model.

We present an individual-based experimental framework to identify and estimate the main parameters governing bacterial conjugation at the individual cell scale. From this analysis, we have established that transient periods of unregulated plasmid transfer within newly formed transconjugant cells, together with contact mechanics arising from cellular growth and division, are the two main processes determining the emergent inability of the pWW0 TOL plasmid to fully invade spatially structured Pseudomonas putida populations. We have also shown that pWW0 conjugation occurs mainly at advanced stages of the growth cycle and that nongrowing cells, even when exposed to high nutrient concentrations, do not display conjugal activity. These results do not support previous hypotheses relating conjugation decay in the deeper cell layers of bacterial biofilms to nutrient depletion and low physiological activity. We observe, however, that transient periods of elevated plasmid transfer in newly formed transconjugant cells are offset by unfavorable cell-to-cell contact mechanics, which ultimately precludes the pWWO TOL plasmid from fully invading tightly packed multicellular P. putida populations such as microcolonies and biofilms.

Adherent growth of Pseudomonas putida KT2440 with and without the TOL plasmid (pWWO) at the solid-liquid and air-liquid interface was examined. We compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal laser scanning microscopy. The TOL-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. Microscopy using fluorescent nucleic acid-specific stains revealed differences in the production of extracellular polymeric substances: TOL carriage leads to more extracellular DNA (eDNA) in pellicles and biofilms. Pellicles were dissolved by DNase I treatment. Enhanced cell lysis due to plasmid carriage was ruled out as the mechanism for eDNA release. We report, for the first time, that carriage of a conjugative plasmid leads to increased biofilm formation by production of eDNA.

One-stage autotrophic nitrogen (N) removal, requiring the simultaneous activity of aerobic and anaerobic ammonium oxidizing bacteria (AOB and AnAOB), can be obtained in spatially redox-stratified biofilms. However, previous experience with Membrane-Aerated Biofilm Reactors (MABRs) has revealed a difficulty in reducing the abundance and activity of nitrite oxidizing bacteria (NOB), which drastically lowers process efficiency. Here we show how sequential aeration is an effective strategy to attain autotrophic N removal in MABRs: Two separate MABRs, which displayed limited or no N removal under continuous aeration, could remove more than 5.5 g N/m(2)/day (at loads up to 8 g N/m(2)/day) by controlled variation of sequential aeration regimes. Daily averaged ratios of the surficial loads of O(2) (oxygen) to NH(4)(+) (ammonium) (L(O(2))/L(NH(4))) were close to 1.73 at this optimum. Real-time quantitative PCR based on 16S rRNA gene confirmed that sequential aeration, even at elevated average O(2) loads, stimulated the abundance of AnAOB and AOB and prevented the increase in NOB. Nitrous oxide (N(2)O) emissions were 100-fold lower compared to other anaerobic ammonium oxidation (Anammox)-nitritation systems. Hence, by applying periodic aeration to MABRs, one-stage autotrophic N removal biofilm reactors can be easily obtained, displaying very competitive removal rates, and negligible N(2)O emissions.

Flagellar motility, a mode of active motion shared by many prokaryotic species, is recognized as a key mechanism enabling population dispersal and resource acquisition in microbial communities living in marine, freshwater, and other liquid-replete habitats. By contrast, its role in variably hydrated habitats, where water dynamics result in fragmented aquatic habitats connected by micrometric films, is debated. Here, we quantify the spatial dynamics of Pseudomonas putida KT2440 and its nonflagellated isogenic mutant as affected by the hydration status of a rough porous surface using an experimental system that mimics aquatic habitats found in unsaturated soils. The flagellar motility of the model soil bacterium decreased sharply within a small range of water potential (0 to −2 kPa) and nearly ceased in liquid films of effective thickness smaller than 1.5 μm. However, bacteria could rapidly resume motility in response to periodic increases in hydration. We propose a biophysical model that captures key effects of hydration and liquid-film thickness on individual cell velocity and use a simple roughness network model to simulate colony expansion. Model predictions match experimental results reasonably well, highlighting the role of viscous and capillary pinning forces in hindering flagellar motility. Although flagellar motility seems to be restricted to a narrow range of very wet conditions, fitness gains conferred by fast surface colonization during transient favorable periods might offset the costs associated with flagella synthesis and explain the sustained presence of flagellated prokaryotes in partially saturated habitats such as soil surfaces.

Despite their role in soil functioning, the ecology of nitrite-oxidizing bacteria, NOB, and their response to disturbances such as those generated by agricultural practices are scarcely known. Over the course of 17 months, we surveyed the potential nitrite oxidation, PNO, the abundance of the Nitrobacter- and Nitrospira-like NOB (by quantitative PCR) and the community structure of the Nitrobacter-like NOB (by PCR-DGGE and cloning-sequencing targeting the nxrA gene) in soils for four treatments: after establishment of tillage on a previously no-tillage system, after cessation of tillage on a previously tillage system, and on control tillage and no-tillage systems. Key soil variables (moisture, organic carbon content and gross mineralization--i.e. ammonification--measured by the 15N dilution technique) were also surveyed. PNO was always higher for the no-tillage than tillage treatments. Establishment of tillage led to a strong and rapid decrease in PNO whereas cessation of tillage did not change PNO even after 17 months. PNO was strongly and positively correlated to the abundance of Nitrobacter-like NOB and was also strongly related to gross mineralization, a proxy of N-availability; in contrast, PNO was weakly and negatively correlated to the abundance of Nitrospira-like NOB. Selection of a dominant population was observed under no-tillage, and PNO was loosely correlated to the community structure of Nitrobacter-like NOB. Our results demonstrate that Nitrobacter-like NOB are the key functional players within the NOB community in soils with high N availability and high activity level, and that changes in PNO are due to shifts between Nitrospira-like and Nitrobacter-like NOB and to a weaker extent by shifts of populations within Nitrobacter-like NOB.

Discharge of nitrogenous components to water bodies can cause eutrophication, deterioration of water quality, toxicity to aquatic life, and pose a potential hazard to human and animal health. Biological nitrogen removal can remove nitrogenous components via conversion to harmless nitrogen gas with high efficiency and relative low costs. However, the removal of nitrogen from domestic wastewater with a low carbon/nitrogen (C/N) ratio can often be limited in municipal wastewater plants (WWTPs) because organic carbon is a limiting factor for denitrification. The present work reviews innovative bacterial nitrogen removal pathways such as shortcut nitrification/denitrification, simultaneous nitrification/denitrification, and the nitritation Anammox process, which can remove nitrogen with low or zero dosage of organic carbon sources. We conclude that advanced process control and some new biological treatment processes including the modified anaerobic/anoxic/oxic (A2/O) process, the step-feed multistage ...

We examined the presence of iron-oxidizing bacteria (IOB) at a groundwater surface water interface (GSI) impacted by reduced groundwater originating as leachate from an upgradient landfill. IOB enrichments and quantifications were obtained, at high vertical resolution, by an iron/oxygen opposing gradient cultivation method. The depth-resolved soil distribution profiles of water content, Fe(2+), and total Fe indicated sharp gradients within the top 10 cm sediments of the GSI, where the IOB density was the highest. In addition, the vertical distribution of iron-reducing bacteria at the same sampling site mirrored the IOB distribution. Clone libraries from two separate IOB enrichments indicated a stratified IOB community with clear differences at short vertical distances. Alpha- and Betaproteobacteria were the dominant phylotypes. Clones from the near-surface sediment (1-2 cm below ground surface) were dominated by members of the Bradyrhizobiaceae and Comamonadaceae; clones from the deeper sediments were phylogenetically more diverse, dominated by members of the Rhodocyclaceae. The iron deposition profiles indicated that active iron oxidation occurred only within the near-to-surface GSI sediments. The match between the iron deposition profiles and the IOB abundance profiles strongly hints at the contribution of the IOB community to Fe oxidation in this Fe-rich GSI ecosystem.

A mechanistic oxygen transfer model was developed and applied to a flow-through hollow-fiber membrane-aerated biofilm reactor. Model results are compared to conventional clean water test results as well as performance data obtained when an actively nitrifying biofilm was present on the fibers. With the biofilm present, oxygen transfer efficiencies between 30 and 55% were calculated from the measured data including the outlet gas oxygen concentration, ammonia consumption stoichiometry, and oxidized nitrogen production stoichiometry, all of which were in reasonable agreement. The mechanistic model overpredicted the oxygen transfer by a factor of 1.3 relative to the result calculated from the outlet gas oxygen concentration, which was considered the most accurate of the measured benchmarks. A mass transfer coefficient derived from the clean water testing with oxygen sensors at the membrane-liquid interface was the most accurate of the predictive models (overpredicted by a factor of 1.1) while a coefficient determined by measuring bulk liquid dissolved oxygen underpredicted the oxygen transfer by a factor of 3. The mechanistic model was found to be an adequate tool for design because it used the published diffusion and partition coefficients rather than requiring small-scale testing to determine the system-specific mass transfer coefficients.

The physical transport of Escherichia coli in terrestrial environments may require control to prevent its dissemination from potential high-density sources, such as confined animal feedlot operations. Biobarriers, wherein convective flows carrying pathogens pass through a porous matrix with high retentive capacity, may present one such approach. Eight environmental E. coli isolates were selected to conduct operational retention tests (ORT) with potential biobarrier materials Pyrax or dolomite, or silica glass as control. The conditions in the ORT were chosen to simulate conditioning by manure solutes, a pulse application of a bacterial load followed by rainfall infiltration, and natural drainage. Removal was limited, and likely caused by the relatively high velocities during drainage, and the conditioning of otherwise favorable adhesion sites. Flagella-mediated motility showed the strongest correlation to biobarrier retention. Significant variability was observed across the E. coli isolates, but consistently higher retention was observed for cells with external versus intestinal pregrowth histories. E. coli O157:H7 was retained the least with all examined matrices, while E. coli K-12 displayed moderate retention and may not serve as representative model strain. Pyrax is a good candidate biobarrier material given its superior removal ability across the tested E. coli strains.

Soils are known to support a great bacterial diversity down to the millimeter scale, but the mechanisms by which such a large diversity is sustained are largely unknown. A feature of unsaturated soils is that water usually forms thin, poorly-connected films, which limit solute diffusive fluxes. It has been proposed, but never unambiguously experimentally tested, that a low substrate diffusive flux would impact bacterial diversity, by promoting the coexistence between slow-growing bacteria and their potentially faster-growing competitors. We used a simple experimental system, based on a Petri dish and a perforated Teflon® membrane to control diffusive fluxes of substrate (benzoate) whilst permitting direct observation of bacterial colonies. The system was inoculated with prescribed strains of Pseudomonas, whose growth was quantified by microscopic monitoring of the fluorescent proteins they produced. We observed that substrate diffusion limitation reduced the growth rate of the otherwise fast-growing Pseudomonas putida KT2440 strain. This strain out-competed Pseudomonas fluorescens F113 in liquid culture, but its competitive advantage was less marked on solid media, and even disappeared under conditions of low substrate diffusion. Low diffusive fluxes of substrate, characteristic of many unsaturated media (e.g. soils, food products), can thus promote bacterial coexistence in a competitive situation between two strains. This mechanism might therefore contribute to maintaining the noncompetitive diversity pattern observed in unsaturated soils.

A 1-d multi-population biofilm model was constructed to study the effect of heterotrophic activity on completely autotrophic ammonium (NH4+) removal in membrane-aerated (counter-diffusion) versus conventional biofilm systems (co-diffusion). Growth of heterotrophic bacteria (HB) was supported either solely by biomass decay products or by organic carbon (as chemical oxygen demand (COD)) in the influent. Three scenarios were considered: influence of HB growing on biomass decay products on steady-state performance (total nitrogen (TN) removal efficiency); influence of the influent COD/N ratio on steady-state performance (supplying COD in the influent); and impact of dynamic changes in the influent COD/N ratio on TN removal efficiency. The results revealed that the TN removal efficiency in the counter-diffusion biofilm was significantly different when HB were included in the simulations at NH4+ surface loads of LNH4>2.7 g - N m(-2) d(-1). Influent COD significantly altered the microbial community composition in the counter-diffusion biofilm and anaerobic NH4+ oxidation could not be sustained at COD/N>2. The co-diffusion system, however, was less affected and more than 50% of the TN removal originated from anaerobic NH4+ oxidation at those ratios. Perturbation experiments showed that step increases to influent COD/N ratios of 2 or higher over a period of 50 d or longer caused a loss of anaerobic NH4+ oxidation capacity which could not be regained within a reasonable time frame (>1000 d) in the counter-diffusion system. In contrast, simulating a 1-d sloughing event only caused a disturbance of 200 d although a maximum biofilm loss of 90-95% occurred. These results clearly indicate the importance of heterotrophic activity in autotrophic N removal biofilms, especially in counter-diffusion systems where they may compromise N removal capacity.