Metagenomic evidence of a novel family of anammox bacteria in a subsea environment.

Bacteria in the order 'Candidatus Brocadiales' within the phylum Planctomycetes (Planctomycetota) have the remarkable ability to perform anaerobic ammonium oxidation (anammox). Two families of anammox bacteria with different biogeographical distributions have been reported, marine Ca. Scalinduaceae and freshwater Ca. Brocadiaceae. Here we report evidence of three new species within a novel genus and family of anammox bacteria, which were discovered in biofilms of a subsea road tunnel under a fjord in Norway. In this particular ecosystem, the nitrogen cycle is likely fuelled by ammonia from organic matter degradation in the fjord sediments and the rock mass above the tunnel, resulting in the growth of biofilms where anammox bacteria can thrive under oxygen limitation. We resolved several metagenome-assembled genomes (MAGs) of anammox bacteria, including three Ca. Brocadiales MAGs that could not be classified at the family level. MAGs of this novel family had all the diagnostic genes for a full anaerobic ammonium oxidation pathway in which nitrite was probably reduced by a NirK-like reductase. A survey of published molecular data indicated that this new family of anammox bacteria occurs in many marine sediments, where its members presumably would contribute to nitrogen loss.

Combined Deterministic and Stochastic Processes Control Microbial Succession in Replicate Granular Biofilm Reactors.

Granular sludge is an efficient and compact biofilm process for wastewater treatment. However, the ecological factors involved in microbial community assembly during the granular biofilm formation are poorly understood, and little is known about the reproducibility of the process. Here, three replicate bioreactors were used to investigate microbial succession during the formation of granular biofilms. We identified three successional phases. During the initial phase, the successional turnover was high and α-diversity decreased as a result of the selection of taxa adapted to grow on acetate and form aggregates. Despite these dynamic changes, the microbial communities in the replicate reactors were similar. The second successional phase occurred when the settling time was rapidly decreased to selectively retain granules in the reactors. The influence of stochasticity on succession increased and new niches were created as granules emerged, resulting in temporarily increased α-diversity. The third successional phase occurred when the settling time was kept stable and granules dominated the biomass. Turnover was low, and selection resulted in the same abundant taxa in the reactors, but drift, which mostly affected low-abundant community members, caused the community in one reactor to diverge from the other two. Even so, performance was stable and similar between reactors.

Effect of Start-Up Strategies and Electrode Materials on Carbon Dioxide Reduction on Biocathodes.

The enrichment of CO2-reducing microbial biocathodes is challenging. Previous research has shown that a promising approach could be to first enrich bioanodes and then lower the potential so the electrodes are converted into biocathodes. However, the effect of such a transition on the microbial community on the electrode has not been studied. The goal of this study was thus to compare the start-up of biocathodes from preenriched anodes with direct start-up from bare electrodes and to investigate changes in microbial community composition. The effect of three electrode materials on the long-term performance of the biocathodes was also investigated. In this study, preenrichment of acetate-oxidizing bioanodes did not facilitate the start-up of biocathodes. It took about 170 days for the preenriched electrodes to generate substantial cathodic current, compared to 83 days for the bare electrodes. Graphite foil and carbon felt cathodes produced higher current at the beginning of the experiment than did graphite rods. However, all electrodes produced similar current densities at the end of the over 1-year-long study (2.5 A/m2). Methane was the only product detected during operation of the biocathodes. Acetate was the only product detected after inhibition of the methanogens. Microbial community analysis showed that Geobacter sp. dominated the bioanodes. On the biocathodes, the Geobacter sp. was succeeded by Methanobacterium spp., which made up more than 80% of the population. After inhibition of the methanogens, Acetobacterium sp. became dominant on the electrodes (40% relative abundance). The results suggested that bioelectrochemically generated H2 acted as an electron donor for CO2 reduction.IMPORTANCE In microbial electrochemical systems, living microorganisms function as catalysts for reactions on the anode and/or the cathode. There is a variety of potential applications, ranging from wastewater treatment and biogas generation to production of chemicals. Systems with biocathodes could be used to reduce CO2 to methane, acetate, or other high-value chemicals. The technique can be used to convert solar energy to chemicals. However, enriching biocathodes that are capable of CO2 reduction is more difficult and less studied than enriching bioanodes. The effect of different start-up strategies and electrode materials on the microbial communities that are enriched on biocathodes has not been studied. The purpose of this study was to investigate two different start-up strategies and three different electrode materials for start-up and long-term operation of biocathodes capable of reducing CO2 to valuable biochemicals.

The mechanisms of granulation of activated sludge in wastewater treatment, its optimization, and impact on effluent quality.

Granular activated sludge has gained increasing interest due to its potential in treating wastewater in a compact and efficient way. It is well-established that activated sludge can form granules under certain environmental conditions such as batch-wise operation with feast-famine feeding, high hydrodynamic shear forces, and short settling time which select for dense microbial aggregates. Aerobic granules with stable structure and functionality have been obtained with a range of different wastewaters seeded with different sources of sludge at different operational conditions, but the microbial communities developed differed substantially. In spite of this, granule instability occurs. In this review, the available literature on the mechanisms involved in granulation and how it affects the effluent quality is assessed with special attention given to the microbial interactions involved. To be able to optimize the process further, more knowledge is needed regarding the influence of microbial communities and their metabolism on granule stability and functionality. Studies performed at conditions similar to full-scale such as fluctuation in organic loading rate, hydrodynamic conditions, temperature, incoming particles, and feed water microorganisms need further investigations.

Nonoxidative removal of organics in the activated sludge process.

The activated sludge process is commonly used to treat wastewater by aerobic oxidation of organic pollutants into carbon dioxide and water. However, several nonoxidative mechanisms can also contribute to removal of organics. Sorption onto activated sludge can remove a large fraction of the colloidal and particulate wastewater organics. Intracellular storage of, e.g., polyhydroxyalkanoates (PHA), triacylglycerides (TAG), or wax esters can convert wastewater organics into precursors for high-value products. Recently, several environmental, economic, and technological drivers have stimulated research on nonoxidative removal of organics for wastewater treatment. In this paper, we review these nonoxidative removal mechanisms as well as the existing and emerging process configurations that make use of them for wastewater treatment. Better utilization of nonoxidative processes in activated sludge could reduce the wasteful aerobic oxidation of organic compounds and lead to more resource-efficient wastewater treatment plants.

Incorporating expert judgments in utility evaluation of bacteroidales qPCR assays for microbial source tracking in a drinking water source.

Several assays for the detection of host-specific genetic markers of the order Bacteroidales have been developed and used for microbial source tracking (MST) in environmental waters. It is recognized that the source-sensitivity and source-specificity are unknown and variable when introducing these assays in new geographic regions, which reduces their reliability and use. A Bayesian approach was developed to incorporate expert judgments with regional assay sensitivity and specificity assessments in a utility evaluation of a human and a ruminant-specific qPCR assay for MST in a drinking water source. Water samples from Lake Rådasjön were analyzed for E. coli, intestinal enterococci and somatic coliphages through cultivation and for human (BacH) and ruminant-specific (BacR) markers through qPCR assays. Expert judgments were collected regarding the probability of human and ruminant fecal contamination based on fecal indicator organism data and subjective information. Using Bayes formula, the conditional probability of a true human or ruminant fecal contamination given the presence of BacH or BacR was determined stochastically from expert judgments and regional qPCR assay performance, using Beta distributions to represent uncertainties. A web-based computational tool was developed for the procedure, which provides a measure of confidence to findings of host-specific markers and demonstrates the information value from these assays.

Tools for T-RFLP data analysis using Excel.

BACKGROUND: Terminal restriction fragment length polymorphism (T-RFLP) analysis is a DNA-fingerprinting method that can be used for comparisons of the microbial community composition in a large number of samples. There is no consensus on how T-RFLP data should be treated and analyzed before comparisons between samples are made, and several different approaches have been proposed in the literature. The analysis of T-RFLP data can be cumbersome and time-consuming, and for large datasets manual data analysis is not feasible. The currently available tools for automated T-RFLP analysis, although valuable, offer little flexibility, and few, if any, options regarding what methods to use. To enable comparisons and combinations of different data treatment methods an analysis template and an extensive collection of macros for T-RFLP data analysis using Microsoft Excel were developed. RESULTS: The Tools for T-RFLP data analysis template provides procedures for the analysis of large T-RFLP datasets including application of a noise baseline threshold and setting of the analysis range, normalization and alignment of replicate profiles, generation of consensus profiles, normalization and alignment of consensus profiles and final analysis of the samples including calculation of association coefficients and diversity index. The procedures are designed so that in all analysis steps, from the initial preparation of the data to the final comparison of the samples, there are various different options available. The parameters regarding analysis range, noise baseline, T-RF alignment and generation of consensus profiles are all given by the user and several different methods are available for normalization of the T-RF profiles. In each step, the user can also choose to base the calculations on either peak height data or peak area data. CONCLUSIONS: The Tools for T-RFLP data analysis template enables an objective and flexible analysis of large T-RFLP datasets in a widely used spreadsheet application.

Impact of T-RFLP data analysis choices on assessments of microbial community structure and dynamics.

BACKGROUND: Terminal restriction fragment length polymorphism (T-RFLP) analysis is a common DNA-fingerprinting technique used for comparisons of complex microbial communities. Although the technique is well established there is no consensus on how to treat T-RFLP data to achieve the highest possible accuracy and reproducibility. This study focused on two critical steps in the T-RFLP data treatment: the alignment of the terminal restriction fragments (T-RFs), which enables comparisons of samples, and the normalization of T-RF profiles, which adjusts for differences in signal strength, total fluorescence, between samples. RESULTS: Variations in the estimation of T-RF sizes were observed and these variations were found to affect the alignment of the T-RFs. A novel method was developed which improved the alignment by adjusting for systematic shifts in the T-RF size estimations between the T-RF profiles. Differences in total fluorescence were shown to be caused by differences in sample concentration and by the gel loading. Five normalization methods were evaluated and the total fluorescence normalization procedure based on peak height data was found to increase the similarity between replicate profiles the most. A high peak detection threshold, alignment correction, normalization and the use of consensus profiles instead of single profiles increased the similarity of replicate T-RF profiles, i.e. lead to an increased reproducibility. The impact of different treatment methods on the outcome of subsequent analyses of T-RFLP data was evaluated using a dataset from a longitudinal study of the bacterial community in an activated sludge wastewater treatment plant. Whether the alignment was corrected or not and if and how the T-RF profiles were normalized had a substantial impact on ordination analyses, assessments of bacterial dynamics and analyses of correlations with environmental parameters. CONCLUSIONS: A novel method for the evaluation and correction of the alignment of T-RF profiles was shown to reduce the uncertainty and ambiguity in alignments of T-RF profiles. Large differences in the outcome of assessments of bacterial community structure and dynamics were observed between different alignment and normalization methods. The results of this study can therefore be of value when considering what methods to use in the analysis of T-RFLP data.

Three-dimensional stratification of bacterial biofilm populations in a moving bed biofilm reactor for nitritation-anammox.

Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers.

Biofilm colonization and succession in a full-scale partial nitritation-anammox moving bed biofilm reactor.

BACKGROUND: Partial nitritation-anammox (PNA) is a biological nitrogen removal process commonly used in wastewater treatment plants for the treatment of warm and nitrogen-rich sludge liquor from anaerobic digestion, often referred to as sidestream wastewater. In these systems, biofilms are frequently used to retain biomass with aerobic ammonia-oxidizing bacteria (AOB) and anammox bacteria, which together convert ammonium to nitrogen gas. Little is known about how these biofilm communities develop, and whether knowledge about the assembly of biofilms in natural communities can be applied to PNA biofilms. RESULTS: We followed the start-up of a full-scale PNA moving bed biofilm reactor for 175 days using shotgun metagenomics. Environmental filtering likely restricted initial biofilm colonization, resulting in low phylogenetic diversity, with the initial microbial community comprised mainly of Proteobacteria. Facilitative priority effects allowed further biofilm colonization, with the growth of initial aerobic colonizers promoting the arrival and growth of anaerobic taxa like methanogens and anammox bacteria. Among the early colonizers were known 'oligotrophic' ammonia oxidizers including comammox Nitrospira and Nitrosomonas cluster 6a AOB. Increasing the nitrogen load in the bioreactor allowed colonization by 'copiotrophic' Nitrosomonas cluster 7 AOB and resulted in the exclusion of the initial ammonia- and nitrite oxidizers. CONCLUSIONS: We show that complex dynamic processes occur in PNA microbial communities before a stable bioreactor process is achieved. The results of this study not only contribute to our knowledge about biofilm assembly and PNA bioreactor start-up but could also help guide strategies for the successful implementation of PNA bioreactors. Video Abstract.

New methods for analysis of spatial distribution and coaggregation of microbial populations in complex biofilms.

In biofilms, microbial activities form gradients of substrates and electron acceptors, creating a complex landscape of microhabitats, often resulting in structured localization of the microbial populations present. To understand the dynamic interplay between and within these populations, quantitative measurements and statistical analysis of their localization patterns within the biofilms are necessary, and adequate automated tools for such analyses are needed. We have designed and applied new methods for fluorescence in situ hybridization (FISH) and digital image analysis of directionally dependent (anisotropic) multispecies biofilms. A sequential-FISH approach allowed multiple populations to be detected in a biofilm sample. This was combined with an automated tool for vertical-distribution analysis by generating in silico biofilm slices and the recently developed Inflate algorithm for coaggregation analysis of microbial populations in anisotropic biofilms. As a proof of principle, we show distinct stratification patterns of the ammonia oxidizers Nitrosomonas oligotropha subclusters I and II and the nitrite oxidizer Nitrospira sublineage I in three different types of wastewater biofilms, suggesting niche differentiation between the N. oligotropha subclusters, which could explain their coexistence in the same biofilms. Coaggregation analysis showed that N. oligotropha subcluster II aggregated closer to Nitrospira than did N. oligotropha subcluster I in a pilot plant nitrifying trickling filter (NTF) and a moving-bed biofilm reactor (MBBR), but not in a full-scale NTF, indicating important ecophysiological differences between these phylogenetically closely related subclusters. By using high-resolution quantitative methods applicable to any multispecies biofilm in general, the ecological interactions of these complex ecosystems can be understood in more detail.

Short-term microbial release during rain events from on-site sewers and cattle in a surface water source.

The protection of drinking water from pathogens such as Cryptosporidium and Giardia requires an understanding of the short-term microbial release from faecal contamination sources in the catchment. Flow-weighted samples were collected during two rainfall events in a stream draining an area with on-site sewers and during two rainfall events in surface runoff from a bovine cattle pasture. Samples were analysed for human (BacH) and ruminant (BacR) Bacteroidales genetic markers through quantitative polymerase chain reaction (qPCR) and for sorbitol-fermenting bifidobacteria through culturing as a complement to traditional faecal indicator bacteria, somatic coliphages and the parasitic protozoa Cryptosporidium spp. and Giardia spp. analysed by standard methods. Significant positive correlations were observed between BacH, Escherichia coli, intestinal enterococci, sulphite-reducing Clostridia, turbidity, conductivity and UV254 in the stream contaminated by on-site sewers. For the cattle pasture, no correlation was found between any of the genetic markers and the other parameters. Although parasitic protozoa were not detected, the analysis for genetic markers provided baseline data on the short-term faecal contamination due to these potential sources of parasites. Background levels of BacH and BacR makers in soil emphasise the need to including soil reference samples in qPCR-based analyses for Bacteroidales genetic markers.

Resistance of aerobic granular sludge microbiomes to periodic loss of biomass.

Granular sludge is a biofilm process used for wastewater treatment which is currently being implemented worldwide. It is important to understand how disturbances affect the microbial community and performance of reactors. Here, two acetate-fed replicate reactors were inoculated with acclimatized sludge and the reactor performance, and the granular sludge microbial community succession were studied for 149 days. During this time, the microbial community was challenged by periodically removing half of the reactor biomass, subsequently increasing the food-to-microorganism (F/M) ratio. Diversity analysis together with null models show that overall, the microbial communities were resistant to the disturbances, observing some minor effects on polyphosphate-accumulating and denitrifying microbial communities and their associated reactor functions. Community turnover was driven by drift and random granule loss, and stochasticity was the governing ecological process for community assembly. These results evidence the aerobic granular sludge process as a robust system for wastewater treatment.

Diversity and dynamics of Archaea in an activated sludge wastewater treatment plant.

BACKGROUND: The activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. While the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. The diversity and dynamics of the Archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes. RESULTS: The Archaea community was dominated by Methanosaeta-like species. During a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. Water temperature appeared to be the process parameter that affected the community composition the most. Several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. The Archaea were estimated to make up 1.6% of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes. CONCLUSIONS: The results presented here show that Archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include Archaea in future studies of microbial communities in activated sludge.

Decay of Bacteroidales genetic markers in relation to traditional fecal indicators for water quality modeling of drinking water sources.

The implementation of microbial fecal source tracking (MST) methods in drinking water management is limited by the lack of knowledge on the transport and decay of host-specific genetic markers in water sources. To address these limitations, the decay and transport of human (BacH) and ruminant (BacR) fecal Bacteroidales 16S rRNA genetic markers in a drinking water source (Lake Rådasjön in Sweden) were simulated using a microbiological model coupled to a three-dimensional hydrodynamic model. The microbiological model was calibrated using data from outdoor microcosm trials performed in March, August, and November 2010 to determine the decay of BacH and BacR markers in relation to traditional fecal indicators. The microcosm trials indicated that the persistence of BacH and BacR in the microcosms was not significantly different from the persistence of traditional fecal indicators. The modeling of BacH and BacR transport within the lake illustrated that the highest levels of genetic markers at the raw water intakes were associated with human fecal sources (on-site sewers and emergency sewer overflow). This novel modeling approach improves the interpretation of MST data, especially when fecal pollution from the same host group is released into the water source from different sites in the catchment.

Estimation of pathogen concentrations in a drinking water source using hydrodynamic modelling and microbial source tracking.

The faecal contamination of drinking water sources can lead to waterborne disease outbreaks. To estimate a potential risk for waterborne infections caused by faecal contamination of drinking water sources, knowledge of the pathogen concentrations in raw water is required. We suggest a novel approach to estimate pathogen concentrations in a drinking water source by using microbial source tracking data and fate and transport modelling. First, the pathogen (norovirus, Cryptosporidium, Escherichia coli O157/H7) concentrations in faecal contamination sources around the drinking water source Lake Rådasjön in Sweden were estimated for endemic and epidemic conditions using measured concentrations of faecal indicators (E. coli and Bacteroidales genetic markers). Afterwards, the fate and transport of pathogens within the lake were simulated using a three-dimensional coupled hydrodynamic and microbiological model. This approach provided information on the contribution from different contamination sources to the pathogen concentrations at the water intake of a drinking water treatment plant. This approach addresses the limitations of monitoring and provides data for quantitative microbial risk assessment (QMRA) and risk management in the context of faecal contamination of surface drinking water sources.

Disturbance-based management of ecosystem services and disservices in partial nitritation-anammox biofilms.

The resistance and resilience provided by functional redundancy, a common feature of microbial communities, is not always advantageous. An example is nitrite oxidation in partial nitritation-anammox (PNA) reactors designed for nitrogen removal in wastewater treatment, where suppression of nitrite oxidizers like Nitrospira is sought. In these ecosystems, biofilms provide microhabitats with oxygen gradients, allowing the coexistence of aerobic and anaerobic bacteria. We designed a disturbance experiment where PNA biofilms, treating water from a high-rate activated sludge process, were constantly or intermittently exposed to anaerobic sidestream wastewater, which has been proposed to inhibit nitrite oxidizers. With increasing sidestream exposure we observed decreased abundance, alpha-diversity, functional versatility, and hence functional redundancy, among Nitrospira in the PNA biofilms, while the opposite patterns were observed for anammox bacteria within Brocadia. At the same time, species turnover was observed for aerobic ammonia-oxidizing Nitrosomonas populations. The different exposure regimens were associated with metagenomic assembled genomes of Nitrosomonas, Nitrospira, and Brocadia, encoding genes related to N-cycling, substrate usage, and osmotic stress response, possibly explaining the three different patterns by niche differentiation. These findings imply that disturbances can be used to manage the functional redundancy of biofilm microbiomes in a desirable direction, which should be considered when designing operational strategies for wastewater treatment.

Ecological role of a seaweed secondary metabolite for a colonizing bacterial community.

Bacteria associated with seaweeds can both harm and benefit their hosts. Many seaweed species are known to produce compounds that inhibit growth of bacterial isolates, but the ecological role of seaweed metabolites for the associated bacterial community structure is not well understood. In this study the response of a colonizing bacterial community to the secondary metabolite (1,1,3,3-tetrabromo-2-heptanone) from the red alga Bonnemaisonia hamifera was investigated by using field panels coated with the metabolite at a range of concentrations covering those measured at the algal surface. The seaweed metabolite has previously been shown to have antibacterial effects. The metabolite significantly affected the natural fouling community by (i) altering the composition, (ii) altering the diversity by increasing the evenness and (iii) decreasing the density, as measured by terminal restriction fragment length polymorphism in conjunction with clone libraries of the 16S rRNA genes and by bacterial enumeration. No single major bacterial taxon (phylum, class) was particularly affected by the metabolite. Instead changes in community composition were observed at a more detailed phylogenetic level. This indicates a broad specificity of the seaweed metabolite against bacterial colonization, which is supported by the observation that the bacterial density was significantly affected at a lower concentration (0.02 µg cm⁻²) than the composition (1-2.5 µg cm⁻²) and the evenness (5 µg cm⁻²) of the bacterial communities. Altogether, the results emphasize the role of secondary metabolites for control of the density and structure of seaweed-associated bacterial communities.

Long-term stability of partial nitritation-anammox for treatment of municipal wastewater in a moving bed biofilm reactor pilot system.

Nitrogen removal from the mainstream of municipal wastewater with partial nitritation-anammox (PNA) would be highly beneficial with regard to the uses of energy and organic carbon. However, the challenges of process instability, low nitrogen removal rates (NRR) and unwanted aerobic nitrite oxidation need to be solved to reach large-scale implementation. Here, we have operated pilot-scale moving bed biofilm reactors (MBBRs) for mainstream treatment, together with sidestream treatment of sludge liquor from anaerobic digestors, for over 900 days to investigate process stability, reactor performance and microbial community structure at realistic conditions. The MBBR biofilm contained stable and high relative abundances of anammox bacteria (10-32%) consisting of two major Brocadia sp. populations, and several populations of aerobic ammonia-oxidising bacteria (AOB) within Nitrosomonas sp. (0.2-3.1%), as assessed by 16S rDNA amplicon sequencing. In addition, nitrite-oxidising bacteria (NOB) consisting of Nitrospira sp. (0.4-0.8%) and Nitrotoga sp. (up to 0.4%) were present. Nitrogen was removed at a peak rate of 0.66 g N m-2 d-1 (0.13 kg N m-3 d-1) with a nitrate production over ammonium consumption of 15% by the NOB, at operation with continuous aeration at 15 °C. However, during most periods with continuous aeration, the NRR was lower (≈ 0.45 g N m-2 d-1), with larger relative nitrate production (≈40%), presumably due to problems to maintain stable residual ammonium concentrations during wet-weather mainstream flows. Changing reactor operation to intermittent aeration decreased the NRR but did not help in suppressing the NOB. The study shows that with MBBRs, stable mainstream PNA can be attained at realistic NRR, but with need for post-treatment of nitrate, since effective NOB suppression was hard to achieve.

Correction to: Hill-based dissimilarity indices and null models for analysis of microbial community assembly.

An amendment to this paper has been published and can be accessed via the original article.