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Nitrous oxide (N2 O) is emitted during microbiological nitrogen (N) conversion processes, when N2 O production exceeds N2 O consumption. The magnitude of N2 O production vs. consumption varies with pH and controlling net N2 O production might be feasible by choice of system pH. This article reviews how pH affects enzymes, pathways and microorganisms that are involved in N-conversions in water engineering applications. At a molecular level, pH affects activity of cofactors and structural elements of relevant enzymes by protonation or deprotonation of amino acid residues or solvent ligands, thus causing steric changes in catalytic sites or proton/electron transfer routes that alter the enzymes' overall activity. Augmenting molecular information with, e.g., nitritation or denitrification rates yields explanations of changes in net N2 O production with pH. Ammonia oxidizing bacteria are of highest relevance for N2 O production, while heterotrophic denitrifiers are relevant for N2 O consumption at pH > 7.5. Net N2 O production in N-cycling water engineering systems is predicted to display a 'bell-shaped' curve in the range of pH 6.0-9.0 with a maximum at pH 7.0-7.5. Net N2 O production at acidic pH is dominated by N2 O production, whereas N2 O consumption can outweigh production at alkaline pH. Thus, pH 8.0 may be a favourable pH set-point for water treatment applications regarding net N2 O production.

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.

Exploring the variation in microbial community diversity between locations (β diversity) is a central topic in microbial ecology. Currently, there is no consensus on how to set the significance threshold for β diversity. Here, we describe and quantify the technical components of β diversity, including those associated with the process of subsampling. These components exist for any proposed β diversity measurement procedure. Further, we introduce a strategy to set significance thresholds for β diversity of any group of microbial samples using rarefaction, invoking the notion of a meta-community. The proposed technique was applied to several in silico generated operational taxonomic unit (OTU) libraries and experimental 16S rRNA pyrosequencing libraries. The latter represented microbial communities from different biological rapid sand filters at a full-scale waterworks. We observe that β diversity, after subsampling, is inflated by intra-sample differences; this inflation is avoided in the proposed method. In addition, microbial community evenness (Gini > 0.08) strongly affects all β diversity estimations due to bias associated with rarefaction. Where published methods to test β significance often fail, the proposed meta-community-based estimator is more successful at rejecting insignificant β diversity values. Applying our approach, we reveal the heterogeneous microbial structure of biological rapid sand filters both within and across filters.

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.

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.

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.

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.

Exploring the variation in microbial community diversity between locations (β diversity) is a central topic in microbial ecology. Currently, there is no consensus on how to set the significance threshold for β diversity. Here, we describe and quantify the technical components of β diversity, including those associated with the process of subsampling. These components exist for any proposed β diversity measurement procedure. Further, we introduce a strategy to set significance thresholds for β diversity of any group of microbial samples using rarefaction, invoking the notion of a meta-community. The proposed technique was applied to several in silico generated operational taxonomic unit (OTU) libraries and experimental 16S rRNA pyrosequencing libraries. The latter represented microbial communities from different biological rapid sand filters at a full-scale waterworks. We observe that β diversity, after subsampling, is inflated by intra-sample differences; this inflation is avoided in the proposed method. In addition, microbial community evenness (Gini 0.08) strongly affects all β diversity estimations due to bias associated with rarefaction. Where published methods to test β significance often fail, the proposed meta-community-based estimator is more successful at rejecting insignificant β diversity values. Applying our approach, we reveal the heterogeneous microbial structure of biological rapid sand filters both within and across filters.

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 (relative 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 parameters 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 framework 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.

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.

The link between nitritation success in a membrane-aerated biofilm reactor (MABR) and the composition of the initial ammonia- and nitrite-oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat-sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO(2)(-)-N/NH(4)(+)-N) were obtained in the Inoculum B- (55.2-56.4%) versus the Inoculum A- (20.2-22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100-150 µm), but the AOB profiles [based on 16S rRNA gene targeted real-time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B- versus A-initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A- and B-initiated reactors were Nitrosospira spp. (48.9-61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8-63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell(-1) h(-1) versus 0.79 fmol cell(-1) h(-1) ). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.

The link between nitritation success in a membrane-aerated biofilm reactor (MABR) and the composition of the initial ammonia- and nitrite-oxidizing bacterial (AOB and NOB) population was investigated. Four identically operated flat-sheet type MABRs were initiated with two different inocula: from an autotrophic nitrifying bioreactor (Inoculum A) or from a municipal wastewater treatment plant (Inoculum B). Higher nitritation efficiencies (NO(2)(-)-N/NH(4)(+)-N) were obtained in the Inoculum B- (55.2-56.4%) versus the Inoculum A- (20.2-22.1%) initiated reactors. The biofilms had similar oxygen penetration depths (100-150 µm), but the AOB profiles [based on 16S rRNA gene targeted real-time quantitative PCR (qPCR)] revealed different peak densities at or distant from the membrane surface in the Inoculum B- versus A-initiated reactors, respectively. Quantitative fluorescence in situ hybridization (FISH) revealed that the predominant AOB in the Inoculum A- and B-initiated reactors were Nitrosospira spp. (48.9-61.2%) versus halophilic and halotolerant Nitrosomonas spp. (54.8-63.7%), respectively. The latter biofilm displayed a higher specific AOB activity than the former biofilm (1.65 fmol cell(-1) h(-1) versus 0.79 fmol cell(-1) h(-1) ). These observations suggest that the AOB and NOB population compositions of the inoculum may determine dominant AOB in the MABR biofilm, which in turn affects the degree of attainable nitritation in an MABR.