Gravity-driven sand filters are the dominant groundwater treatment technology for drinking water production. In the past, physicochemical reactions were often assumed to play the main role in the removal of contaminants, but recent breakthroughs showcase the vital role of microorganisms. In this Current Opinion, we thoroughly assess the current understanding of biology in sand filters and explore the potential benefits of shifting toward designs aimed at promoting biological reactions. We highlight the main bottlenecks and propose key areas to be explored toward the next generation of sustainable, resource-efficient groundwater biofilters.@en

Rapid sand filters are established and widely applied technologies for groundwater treatment. In these filters, main groundwater contaminants such as iron, manganese, and ammonium are oxidized and removed. Conventionally, intensive aeration is employed to provide oxygen for these redox reactions. While effective, intensive aeration promotes flocculent iron removal, which results in iron oxide flocs that rapidly clog the filter. In this study, we operated two parallel full-scale sand filters at different aeration intensities to resolve the relative contribution of homogeneous, heterogeneous and biological iron removal pathways, and identify their operational controls. Our results show that mild aeration in the LOW filter (5 mg/L O2, pH 6.9) promoted biological iron removal and enabled iron oxidation at twice the rate compared to the intensively aerated HIGH filter (>10 mg/L O2, pH 7.4). Microscopy images showed distinctive twisted stalk-like iron solids, the biosignatures of Gallionella ferruginea, both in the LOW filter sand coatings as well as in its backwash solids. In accordance, 10 times higher DNA copy numbers of G. ferruginea were found in the LOW filter effluent. Clogging by biogenic iron solids was slower than by chemical iron flocs, resulting in lower backwash frequencies and yielding four times more water per run. Ultimately, our results reveal that biological iron oxidation can be actively controlled and favoured over competing physico-chemical routes. The production of more compact and practically valuable iron oxide solids is of outmost interest. We conclude that, although counterintuitive, slowing down iron oxidation in the water before filtration enables rapid iron removal in the biofilter.@en

Nitrous oxide (N₂O) is a potent greenhouse gas of primarily microbial origin. Oxic and anoxic emissions are commonly ascribed to autotrophic nitrification and heterotrophic denitrification, respectively. Beyond this established dichotomy, we quantitatively show that heterotrophic denitrification can significantly contribute to aerobic nitrogen turnover and N₂O emissions in complex microbiomes exposed to frequent oxic/anoxic transitions. Two planktonic, nitrification-inhibited enrichment cultures were established under continuous organic carbon and nitrate feeding, and cyclic oxygen availability. Over a third of the influent organic substrate was respired with nitrate as electron acceptor at high oxygen concentrations (>6.5 mg/L). N₂O accounted for up to one-quarter of the nitrate reduced under oxic conditions. The enriched microorganisms maintained a constitutive abundance of denitrifying enzymes due to the oxic/anoxic frequencies exceeding their protein turnover—a common scenario in natural and engineered ecosystems. The aerobic denitrification rates are ascribed primarily to the residual activity of anaerobically synthesised enzymes. From an ecological perspective, the selection of organisms capable of sustaining significant denitrifying activity during aeration shows their competitive advantage over other heterotrophs under varying oxygen availabilities. Ultimately, we propose that the contribution of heterotrophic denitrification to aerobic nitrogen turnover and N₂O emissions is currently underestimated in dynamic environments.

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Rapid sand filters (RSF) are an established and widely applied technology for the removal of dissolved iron (Fe²⁺) and ammonium (NH₄⁺) among other contaminants in groundwater treatment. Most often, biological NH₄⁺oxidation is spatially delayed and starts only upon complete Fe²⁺ depletion. However, the mechanism(s) responsible for the inhibition of NH₄⁺oxidation by Fe²⁺ or its oxidation (by)products remains elusive, hindering further process control and optimization. We used batch assays, lab-scale columns, and full-scale filter characterizations to resolve the individual impact of the main Fe²⁺ oxidizing mechanisms and the resulting products on biological NH₄⁺ oxidation. modeling of the obtained datasets allowed to quantitatively assess the hydraulic implications of Fe²⁺ oxidation. Dissolved Fe²⁺ and the reactive oxygen species formed as byproducts during Fe²⁺ oxidation had no direct effect on ammonia oxidation. The Fe³⁺ oxides on the sand grain coating, commonly assumed to be the main cause for inhibited ammonia oxidation, seemed instead to enhance it. modeling allowed to exclude mass transfer limitations induced by accumulation of iron flocs and consequent filter clogging as the cause for delayed ammonia oxidation. We unequivocally identify the inhibition of NH₄⁺oxidizing organisms by the Fe³⁺ flocs generated during Fe²⁺ oxidation as the main cause for the commonly observed spatial delay in ammonia oxidation. The addition of Fe³⁺ flocs inhibited NH₄⁺oxidation both in batch and column tests, and the removal of Fe³⁺ flocs by backwashing completely re-established the NH₄⁺removal capacity, suggesting that the inhibition is reversible. In conclusion, our findings not only identify the iron form that causes the inhibition, albeit the biological mechanism remains to be identified, but also highlight the ecological importance of iron cycling in nitrifying environments.

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Biomarkers such as functional gene mRNA (transcripts) and proteins (enzymes) provide direct proof of metabolic regulation during the reductive dechlorination (RD) of chlorinated ethenes (CEs). Yet, current models to simulate their spatiotemporal variability are not flexible enough to mimic the homologous behavior of RDase functional genes. To this end, we developed new enzyme-based kinetics to model the concentrations of CEs together with the transcript and enzyme levels during RD. First, the model was calibrated to existing microcosm data on RD of cis-DCE. The model mirrored the tceA and vcrA gene expression and the production of their enzymes in Dehalococcoides spp. Considering tceA and vcrA as homologous instead of nonhomologous improved fitting of the mRNA time series. Second, CEs and biomarker patterns were explored as a proof of concept under groundwater flow conditions, considering degraders occurring in immobile and mobile states. Under both microcosm and flow conditions, biomarker-rate relationships were nonlinear hysteretic because tceA and vcrA acted as homologous genes. The mobile biomarkers additionally undergo advective-dispersive transport, which increases the nonlinearity and makes the observed patterns even more challenging to interpret. The model offers a thorough mechanistic description of RD while also allowing simulation of spatiotemporal dynamic patterns of various key biomarkers in aquifers.@en

Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-dependent iron-oxidizing (NDFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with the NDFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new Candidate order, named Siderophiliales. This new species, “Candidatus Siderophilus nitratireducens,” carries genes central genes to iron oxidation (cytochrome c cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). Using thermodynamics, we demonstrate that iron oxidation coupled to nap based dissimilatory reduction of nitrate to nitrite is energetically favorable under realistic Fe3+/Fe2+ and NO3−/NO2− concentration ratios. Ultimately, by bridging the gap between laboratory investigations and nitrate real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NDFOs taxonomic diversity and ecological role.@en

Drinking water treatment plants (DWTPs) are designed to remove physical, chemical, and biological contaminants. However, until recently, the role of DWTPs in minimizing the cycling of antibiotic resistance determinants has got limited attention. In particular, the risk of selecting antibiotic-resistant bacteria (ARB) is largely overlooked in chlorine-free DWTPs where biological processes are applied. Here, we combined high-throughput quantitative PCR and metagenomics to analyze the abundance and dynamics of microbial communities, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) across the treatment trains of two chlorine-free DWTPs involving dune-based and reservoir-based systems. The microbial diversity of the water increased after all biological unit operations, namely rapid and slow sand filtration (SSF), and granular activated carbon filtration. Both DWTPs reduced the concentration of ARGs and MGEs in the water by circa 2.5 log gene copies mL−1, despite their relative increase in the disinfection sub-units (SSF in dune-based and UV treatment in reservoir-based DWTPs). The total microbial concentration was also reduced (2.5 log units), and none of the DWTPs enriched for bacteria containing genes linked to antibiotic resistance. Our findings highlight the effectiveness of chlorine-free DWTPs in supplying safe drinking water while reducing the concentration of antibiotic resistance determinants. To the best of our knowledge, this is the first study that monitors the presence and dynamics of antibiotic resistance determinants in chlorine-free DWTPs.@en

Wastewater treatment plants (WWTPs) are a major source of N₂O, a potent greenhouse gas with 300 times higher global warming potential than CO₂. Several approaches have been proposed for mitigation of N₂O emissions from WWTPs and have shown promising yet only site-specific results. Here, self-sustaining biotrickling filtration, an end-of-the-pipe treatment technology, was tested in situ at a full-scale WWTP under realistic operational conditions. Temporally varying untreated wastewater was used as trickling medium, and no temperature control was applied. The off-gas from the covered WWTP aerated section was conveyed through the pilot-scale reactor, and an average removal efficiency of 57.9 ± 29.1% was achieved during 165 days of operation despite the generally low and largely fluctuating influent N₂O concentrations (ranging between 4.8 and 96.4 ppmv). For the following 60-day period, the continuously operated reactor system removed 43.0 ± 21.2% of the periodically augmented N₂O, exhibiting elimination capacities as high as 5.25 g N₂O m^-3·h^-1. Additionally, the bench-scale experiments performed abreast corroborated the resilience of the system to short-term N₂O starvations. Our results corroborate the feasibility of biotrickling filtration for mitigating N₂O emitted from WWTPs and demonstrate its robustness toward suboptimal field operating conditions and N₂O starvation, as also supported by analyses of the microbial compositions and nosZ gene profiles.

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Rapid sand filters (RSF) are an established and widely applied technology for groundwater treatment. Yet, the underlying interwoven biological and physical-chemical reactions controlling the sequential removal of iron, ammonia and manganese remain poorly understood. To resolve the contribution and interactions between the individual reactions, we studied two full-scale drinking water treatment plant configurations, namely (i) one dual-media (anthracite and quartz sand) filter and (ii) two single-media (quartz sand) filters in series. In situ and ex situ activity tests were combined with mineral coating characterization and metagenome-guided metaproteomics along the depth of each filter. Both plants exhibited comparable performances and process compartmentalization, with most of ammonium and manganese removal occurring only after complete iron depletion. The homogeneity of the media coating and genome-based microbial composition within each compartment highlighted the effect of backwashing, namely the complete vertical mixing of the filter media. In stark contrast to this homogeneity, the removal of the contaminants was strongly stratified within each compartment, and decreased along the filter height. This apparent and longstanding conflict was resolved by quantifying the expressed proteome at different filter heights, revealing a consistent stratification of proteins catalysing ammonia oxidation and protein-based relative abundances of nitrifying genera (up to 2 orders of magnitude difference between top and bottom samples). This implies that microorganisms adapt their protein pool to the available nutrient load at a faster rate than the backwash mixing frequency. Ultimately, these results show the unique and complementary potential of metaproteomics to understand metabolic adaptations and interactions in highly dynamic ecosystems.

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Nitrous oxide (N2O) is a potent greenhouse gas of primarily microbial origin. Aerobic and anoxic emissions are commonly ascribed to nitrification and denitrification, respectively. Beyond this established dichotomy, we quantitatively prove that heterotrophic denitrification can significantly contribute to aerobic nitrogen turnover and N2O emissions in complex microbiomes exposed to frequent oxic/anoxic transitions. Planktonic, nitrification-inhibited denitrifying enrichments respired over a third of the influent organic substrate with nitrate at high oxygen concentrations. N2O accounted for up to one quarter of the aerobically respired nitrate. The constitutive detection of all denitrification enzymes in both anoxic and oxic periods highlight the selective advantage offered by metabolic preparedness in dynamic environments. We posit that aerobic denitrification and associated N2O formation is currently underestimated in dynamic microbial ecosystems.@en

Microorganisms possessing N₂O reductases (NosZ) are the only known environmental sink of N₂O. While oxygen inhibition of NosZ activity is widely known, environments where N₂O reduction occurs are often not devoid of O₂. However, little is known regarding N₂O reduction in microoxic systems. Here, 1.6-L chemostat cultures inoculated with activated sludge samples were sustained for ca. 100 days with low concentration (<2 ppmv) and feed rate (<1.44 µmoles h⁻¹) of N₂O, and the resulting microbial consortia were analyzed via quantitative PCR (qPCR) and metagenomic/metatranscriptomic analyses. Unintended but quantified intrusion of O₂ sustained dissolved oxygen concentration above 4 µM; however, complete N₂O reduction of influent N₂O persisted throughout incubation. Metagenomic investigations indicated that the microbiomes were dominated by an uncultured taxon affiliated to Burkholderiales, and, along with the qPCR results, suggested coexistence of clade I and II N₂O reducers. Contrastingly, metatranscriptomic nosZ pools were dominated by the Dechloromonas-like nosZ subclade, suggesting the importance of the microorganisms possessing this nosZ subclade in reduction of trace N₂O. Further, co-expression of nosZ and ccoNO/cydAB genes found in the metagenome-assembled genomes representing these putative N₂O-reducers implies a survival strategy to maximize utilization of scarcely available electron acceptors in microoxic environmental niches.

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The application of partial nitritation-anammox (PN/A) under mainstream conditions can enable substantial cost savings at wastewater treatment plants (WWTPs), but how process conditions and cell physiology affect anammox performance at psychrophilic temperatures below 15 °C remains poorly understood. We tested 14 anammox communities, including 8 from globally-installed PN/A processes, for (i) specific activity at 10–30 °C, (ii) composition of membrane lipids, and (iii) microbial community structure. We observed that membrane composition and cultivation temperature were closely related to the activity of anammox biomasses. The size of ladderane lipids and the content of bacteriohopanoids were key physiological components related to anammox performance at low temperatures. We also indicate that the adaptation of mesophilic cultures to psychrophilic regime necessitates months, but in some cases can take up to 5 years. Interestingly, biomass enriched in the marine genus “Candidatus Scalindua” displayed outstanding potential for nitrogen removal from cold streams. Collectively, our comprehensive study provides essential knowledge of cold adaptation mechanism, will enable more accurate modelling and suggests highly promising target anammox genera for inoculation and set-up of anammox reactors, in particular for mainstream WWTPs.

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The adaptation of bacteria involved in anaerobic ammonium oxidation (anammox) to low temperatures will enable more efficient removal of nitrogen from sewage across seasons. At lower temperatures, bacteria typically tune the synthesis of their membrane lipids to promote membrane fluidity. However, such adaptation of anammox bacteria lipids, including unique ladderane phospholipids and especially shorter ladderanes with absent phosphatidyl headgroup, is yet to be described in detail. We investigated the membrane lipids composition (UPLC–HRMS/MS) and dominant anammox populations (16S rRNA gene amplicon sequencing, Fluorescence in situ hybridization) in 14 anammox enrichments cultivated at 10–37 °C. “Candidatus Brocadia” appeared to be the dominant organism in all but two laboratory enrichments of “Ca. Scalindua” and “Ca. Kuenenia”. At lower temperatures, the membranes of all anammox populations were composed of shorter [5]-ladderane ester (reduced chain length demonstrated by decreased fraction of C20/(C18 + C20)). This confirmed the previous preliminary evidence on the prominent role of this ladderane fatty acid in low-temperature adaptation. “Ca. Scalindua” and “Ca. Kuenenia” had distinct profile of ladderane lipids compared to “Ca. Brocadia” biomasses with potential implications for adaptability to low temperatures. “Ca. Brocadia” membranes contained a much lower amount of C18 [5]-ladderane esters than reported in the literature for “Ca. Scalindua” at similar temperature and measured here, suggesting that this could be one of the reasons for the dominance of “Ca. Scalindua” in cold marine environments. Furthermore, we propose additional and yet unreported mechanisms for low-temperature adaptation of anammox bacteria, one of which involves ladderanes with absent phosphatidyl headgroup. In sum, we deepen the understanding of cold anammox physiology by providing for the first time a consistent comparison of anammox-based communities across multiple environments.

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This study deals with the effect of aeration control strategies on the nitrogen removal efficiency and nitrous oxide (N₂O) emissions in a partial nitritation–anammox reactor with granular sludge. More specifically, dissolved oxygen (DO) control, constant airflow and effluent ammonium (NH₄⁺) control strategies were compared through a simulation study. Particular attention was paid to the effect of flocs, which are deliberately or unavoidable present besides granules in this type of reactor. When applying DO control, DO setpoints had to be adjusted to the amount of flocs present in the reactor to maintain high nitrogen removal and reduce N₂O emissions, which is difficult to realize in practice because of variable floc fractions. Constant airflow rate control could maintain a good nitrogen removal efficiency independent of the floc fraction in the reactor, but failed in N₂O mitigation. Controlling aeration based on the effluent ammonium concentration results in both high nitrogen removal and relatively low N₂O emissions, also in the presence of flocs. Fluctuations in floc fractions caused significant upsets in nitrogen removal and N₂O emissions under DO control but had less effect at constant airflow and effluent ammonium control. Still, rapid and sharp drops in flocs led to a peak in N₂O emissions at constant airflow and effluent ammonium control. Overall, effluent ammonium control reached the highest average nitrogen removal and lowest N₂O emissions and consumed the lowest aeration energy under fluctuating floc concentrations.

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Partial nitration-anammox is a resource-efficient technology for nitrogen removal from wastewater. However, the advantages of this nitrogen removal technology are challenged by the emission of N₂O, a potent greenhouse gas. In this study, a granular sludge one-stage partial nitritation-anammox reactor comprising granules and flocs was run for 337 days in the presence of influent organics to investigate its effect on N removal and N₂O emissions. Besides, the effect of aeration control strategies and flocs removal was investigated as well. The interpretation of the experimental results was complemented with modelling and simulation. The presence of influent organics (1 g COD g^-1 N) helped to suppress NOB and significantly reduced the overall N₂O emissions while having no significant effect on anammox activity. Besides, long-term monitoring of the reactor indicated that constant airflow rate control resulted in more stable effluent quality and less N₂O emissions than DO control. Still, floc removal reduced N₂O emissions at DO control but increased N₂O emissions at constant airflow rate. Furthermore, anammox bacteria could significantly reduce N₂O production during heterotrophic denitrification, likely via competition for NO with heterotrophs. Overall, this study demonstrated that the presence of influent organics together with proper aeration control strategies and floc management could significantly reduce the N₂O emissions without compromising nitrogen removal efficiency during one-stage partial nitritation-anammox processes.

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The enormous chemical diversity and strain variability of prokaryotic protein glycosylation makes their large-scale exploration exceptionally challenging. Therefore, despite the universal relevance of protein glycosylation across all domains of life, the understanding of their biological significance and the evolutionary forces shaping oligosaccharide structures remains highly limited. Here, we report on a newly established mass binning glycoproteomics approach that establishes the chemical identity of the carbohydrate components and performs untargeted exploration of prokaryotic oligosaccharides from large-scale proteomics data directly. We demonstrate our approach by exploring an enrichment culture of the globally relevant anaerobic ammonium-oxidizing bacterium Ca. Kuenenia stuttgartiensis. By doing so we resolve a remarkable array of oligosaccharides, which are produced by two seemingly unrelated biosynthetic routes, and which modify the same surface-layer protein simultaneously. More intriguingly, the investigated strain also accomplished modulation of highly specialized sugars, supposedly in response to its energy metabolism—the anaerobic oxidation of ammonium—which depends on the acquisition of substrates of opposite charges. Ultimately, we provide a systematic approach for the compositional exploration of prokaryotic protein glycosylation, and reveal a remarkable example for the evolution of complex oligosaccharides in bacteria.

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Hydroxylamine is a key intermediate in several biological reactions of the global nitrogen cycle. However, the role of hydroxylamine in anammox is still not fully understood. In this work, the impact of hydroxylamine (also in combination with other substrates) on the metabolism of a planktonic enrichment culture of the anammox species Ca. Kuenenia stuttgartiensis was studied. Anammox bacteria were observed to produce ammonium both from hydroxylamine and hydrazine, and hydroxylamine was consumed simultaneously with nitrite. Hydrazine accumulation - signature for the presence of anammox bacteria - strongly depended on the available substrates, being higher with ammonium and lower with nitrite. Furthermore, the results presented here indicate that hydrazine accumulation is not the result of the inhibition of hydrazine dehydrogenase, as commonly assumed, but the product of hydroxylamine disproportionation. All kinetic parameters for the identified reactions were estimated by mathematical modelling. Moreover, the simultaneous consumption and growth on ammonium, nitrite and hydroxylamine of anammox bacteria was demonstrated, this was accompanied by a reduction in the nitrate production. Ultimately, this study advances the fundamental understanding of the metabolic versatility of anammox bacteria, and highlights the potential role played by metabolic intermediates (i.e. hydroxylamine, hydrazine) in shaping natural and engineered microbial communities.

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The authors regret that there is an error in one stoichiometric coefficient and in the process rate of NOB in Table 1 of the original publication. The correct table is shown below. The authors would like to apologise for any inconvenience caused.

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A model-based study was developed to analyse the behaviour of Moving Bed Biofilm Reactor (MBBR) and Integrated Fixed-Film Activated Sludge (IFAS) reactor configurations for the removal of nitrogen in the main water line of municipal wastewater treatment plants via partial nitritation/anammox (PN/AMX). The basic principles and underlying mechanisms linking operating conditions to process performance were investigated, with particular focus on nitrite oxidizing bacteria (NOB) repression and resulting volumetric conversion rates. The external mass transfer resistance is a major factor differentiating granular sludge PN/AMX processes from MBBR or IFAS systems. The external mass transfer resistance was found to promote the metabolic coupling between anammox (AMX) and ammonia oxidizing bacteria (AOB), crucial for NOB repression in the biofilm. Operation at low bulk DO prevents NOB proliferation in the flocs of IFAS systems as AMX activity limits nitrite availability (the so-called AMX nitrite sink). Importantly, the effectiveness of the AMX nitrite sink strongly depends on the AMX sensitivity to oxygen. Also, over a broad range of operational conditions, the seeding of AOB from the biofilm played a crucial role in maintaining their activity in the flocs. From a practical perspective, while low DO promotes NOB repression, lower nitrogen loads have to be applied to maintain the same effluent quality. Thus, a trade-off between NOB repression and volumetric conversion capacity needs to be defined. To this end, IFAS allow for higher volumetric rates, but the window of operating conditions with effective NOB repression is smaller than that for MBBR. Ultimately, this study identified the principles controlling NOB in MBBR and IFAS systems and the key differences with granular reactors, allowing for the interpretation of (seemingly contradictory) published experimental results.

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The control of nitrite-oxidizing bacteria (NOB) challenges the implementation of partial nitritation and anammox (PN/A) processes under mainstream conditions. The aim of the present study was to understand how operating conditions impact microbial competition and the control of NOB in hybrid PN/A systems, where biofilm and flocs coexist. A hybrid PN/A moving-bed biofilm reactor (MBBR; also referred to as integrated fixed film activated sludge or IFAS) was operated at 15 °C on aerobically pre-treated municipal wastewater (23 mg _NH4-N L ⁻¹ ). Ammonium-oxidizing bacteria (AOB) and NOB were enriched primarily in the flocs, and anammox bacteria (AMX) in the biofilm. After decreasing the dissolved oxygen concentration (DO) from 1.2 to 0.17 mg _O2 L ⁻¹ - with all other operating conditions unchanged - washout of NOB from the flocs was observed. The activity of the minor NOB fraction remaining in the biofilm was suppressed at low DO. As a result, low effluent NO ₃ ⁻ concentrations (0.5 mg _N L ⁻¹ ) were consistently achieved at aerobic nitrogen removal rates (80 mg _N L ⁻¹ d ⁻¹ ) comparable to those of conventional treatment plants. A simple dynamic mathematical model, assuming perfect biomass segregation with AOB and NOB in the flocs and AMX in the biofilm, was able to qualitatively reproduce the selective washout of NOB from the flocs in response to the decrease in DO-setpoint. Similarly, numerical simulations indicated that flocs removal is an effective operational strategy to achieve the selective washout of NOB. The direct competition for NO ₂ ⁻ between NOB and AMX - the latter retained in the biofilm and acting as a “NO ₂ -sink” - was identified by the model as key mechanism leading to a difference in the actual growth rates of AOB and NOB (i.e., μ _NOB < μ _AOB in flocs) and allowing for the selective NOB washout over a broad range of simulated sludge retention times (SRT = 6.8–24.5 d). Experimental results and model predictions demonstrate the increased operational flexibility, in terms of variables that can be easily controlled by operators, offered by hybrid systems as compared to solely biofilm systems for the control of NOB in mainstream PN/A applications.

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