Application of biological early warning systems in wastewater treatment plants: Introducing a promising approach to monitor changing wastewater composition.

Wastewater treatment plants (WWTPs) are a major source of micropollutants to surface waters. Currently, their chemical or biological monitoring is realized by using grab or composite samples, which provides only snapshots of the current wastewater composition. Especially in WWTPs with industrial input, the wastewater composition can be highly variable and a continuous assessment would be advantageous, but very labor and cost intensive. A promising concept are automated real-time biological early warning systems (BEWS), where living organisms are constantly exposed to the water and an alarm is triggered if the organism's responses exceed a harmful threshold of acute toxicity. Currently, BEWS are established for drinking water and surface water but are seldom applied to monitor wastewater. This study demonstrates that a battery of BEWS using algae (Chlorella vulgaris in the Algae Toximeter, bbe Moldaenke), water flea (Daphnia magna in the DaphTox II, bbe Moldaenke) and gammarids (Gammarus pulex in the Sensaguard, REMONDIS Aqua) can be adapted for wastewater surveillance. For continuous low-maintenance operation, a back-washable membrane filtration system is indispensable for adequate preparation of treated wastewater. Only minor deviations in the reaction of the organisms towards treated and filtered wastewater compared to surface waters were detected. After spiking treated wastewater with two concentrations of the model compounds diuron, chlorpyrifos methyl, and sertraline, the organisms in the different BEWS showed clear responses depending on the respective compound, concentration and mode of action. Immediate effects on photosynthetic activity of algae were detected for diuron exposure, and strong behavioral changes in water flea and gammarids after exposure to chlorpyrifos methyl or sertraline were observed, which triggered automated alarms. Different types of data analysis were applied to extract more information out of the specific behavioral traits, than only provided by the vendors algorithms. To investigate, whether behavioral movement changes can be linked to impact other endpoints, the effects on feeding activity of G. pulex were evaluated and results indicated significant differences between the exposures. Overall, these findings provide an important basis indicating that BEWS have the potential to act as alarm systems for pollution events in the wastewater sector.

Stream microbial communities and ecosystem functioning show complex responses to multiple stressors in wastewater.

Multiple anthropogenic drivers are changing ecosystems globally, with a disproportionate and intensifying impact on freshwater habitats. A major impact of urbanization are inputs from wastewater treatment plants (WWTPs). Initially designed to reduce eutrophication and improve water quality, WWTPs increasingly release a multitude of micropollutants (MPs; i.e., synthetic chemicals) and microbes (including antibiotic-resistant bacteria) to receiving environments. This pollution may have pervasive impacts on biodiversity and ecosystem services. Viewed through multiple lenses of macroecological and ecotoxicological theory, we combined field, flume, and laboratory experiments to determine the effects of wastewater (WW) on microbial communities and organic-matter processing using a standardized decomposition assay. First, we conducted a mensurative experiment sampling 60 locations above and below WWTP discharges in 20 Swiss streams. Microbial respiration and decomposition rates were positively influenced by WW inputs via warming and nutrient enrichment, but with a notable exception: WW decreased the activation energy of decomposition, indicating a "slowing" of this fundamental ecosystem process in response to temperature. Second, next-generation sequencing indicated that microbial community structure below WWTPs was altered, with significant compositional turnover, reduced richness, and evidence of negative MP influences. Third, a series of flume experiments confirmed that although diluted WW generally has positive influences on microbial-mediated processes, the negative effects of MPs are "masked" by nutrient enrichment. Finally, transplant experiments suggested that WW-borne microbes enhance decomposition rates. Taken together, our results affirm the multiple stressor paradigm by showing that different aspects of WW (warming, nutrients, microbes, and MPs) jointly influence ecosystem functioning in complex ways. Increased respiration rates below WWTPs potentially generate ecosystem "disservices" via greater carbon evasion from streams and rivers. However, toxic MP effects may fundamentally alter ecological scaling relationships, indicating the need for a rapprochement between ecotoxicological and macroecological perspectives.

Trends in Micropollutant Biotransformation along a Solids Retention Time Gradient.

For many polar organic micropollutants, biotransformation by activated sludge microorganisms is a major removal process during wastewater treatment. However, our current understanding of how wastewater treatment operations influence microbial communities and their micropollutant biotransformation potential is limited, leaving major parts of observed variability in biotransformation rates across treatment facilities unexplained. Here, we present biotransformation rate constants for 42 micropollutants belonging to different chemical classes along a gradient of solids retention time (SRT). The geometric mean of biomass-normalized first-order rate constants shows a clear increase between 3 and 15 d SRT by 160% and 87%, respectively, in two experiments. However, individual micropollutants show a variety of trends. Rate constants of oxidative biotransformation reactions mostly increased with SRT. Yet, nitrifying activity could be excluded as primary driver. For substances undergoing other than oxidative reactions, i.e., mostly substitution-type reactions, more diverse dependencies on SRT were observed. Most remarkably, characteristic trends were observed for groups of substances undergoing similar types of initial transformation reaction, suggesting that shared enzymes or enzyme systems that are conjointly regulated catalyze biotransformation reactions within such groups. These findings open up opportunities for correlating rate constants with measures of enzyme abundance such as genes or gene products, which in turn should help to identify enzymes associated with the respective biotransformation reactions.

Advancing Biological Wastewater Treatment: Extended Anaerobic Conditions Enhance the Removal of Endocrine and Dioxin-like Activities.

Conventional activated sludge treatment of wastewater does not completely remove micropollutants. Here, extending anaerobic conditions may enhance biodegradation. To explore this, we combined iron-reducing or substrate-limiting and aerobic pilot-scale reactors directly at a wastewater treatment plant. To assess the removal of endocrine disrupting chemicals (EDCs) as group of micropollutants that adversely affects wildlife, we applied a bioanalytical approach. We used in vitro bioassays covering seven receptor-mediated mechanisms of action, including (anti)androgenicity, (anti)estrogenicity, retinoid-like, and dioxin-like activity. Untreated wastewater induced antiandrogenic, estrogenic, antiestrogenic, and retinoid-like activity. Full-scale as well as reactor-scale activated sludge treatment effectively removes the observed effects. Nevertheless, high antiandrogenic and minor dioxin-like and estrogenic effects persisted in the treated effluent that may still be environmentally relevant. The anaerobic post-treatment under substrate-limiting conditions resulted in an additional removal of endocrine activities by 17-40%. The anaerobic pre-treatment under iron-reducing conditions significantly enhanced the removal of the residual effects by 40-75%. In conclusion, this study demonstrates that a further optimization of biological wastewater treatment is possible. Here, implementing iron-reducing anaerobic conditions preceding aerobic treatment appears promising to improve the removal of receptor-mediated toxicity.

Reducing the discharge of micropollutants in the aquatic environment: the benefits of upgrading wastewater treatment plants.

Micropollutants (MPs) as individual compounds or in complex mixtures are relevant for water quality and may trigger unwanted ecological effects. MPs originate from different point and diffuse sources and enter water bodies via different flow paths. Effluents from conventional wastewater treatment plants (WWTPs), in which various MPs are not or not completely removed, is one major source. To improve the water quality and avoid potential negative ecological effects by micropollutants, various measures to reduce the discharge should be taken. In this feature we discuss one of these measures; the benefits of upgrading WWTPs toward reduced MP loads and toxicities from wastewater effluents, using the recently decided Swiss strategy as an example. Based on (i) full-scale case studies using ozonation or powder activated carbon treatment, showing substantial reduction of MP discharges and concomitant reduced toxicities, (ii) social and political acceptance, (iii) technical feasibility and sufficient cost-effectiveness, the Swiss authorities recently decided to implement additional wastewater treatment steps as mitigation strategy to improve water quality. Since MPs are of growing global concern, the concepts and considerations behind the Swiss strategy are explained in this feature, which could be of use for other countries as well. It should be realized that upgrading WWTPs is not the only solution to reduce the discharge of MPs entering the environment, but is part of a broader, multipronged mitigation strategy.

Impact of long-term temperature shifts on activated sludge microbiome dynamics and micropollutant removal.

Micropollutants removal efficiency strongly vary across different aerobic wastewater treatment plants, resulting in their frequent detection in surface and ground waters. Seasonal temperature variation is a major factor influencing plant performance, but it is still unclear how prolonged periods of temperature change impact microbiome and micropollutant biotransformation. This work investigates the effect of long-term temperature variation on the microbial dynamics in an activated sludge system, and the impact on micropollutant biotransformation. Sequencing batch reactors were used as model system and 4-40 °C temperature range was studied. 16S rRNA amplicon sequencing showed that temperature drives microbial structure (gDNA) and activity (RNA), rather than time, and this was stronger below 15 °C and above 25 °C. The microbial community was richest and more diverse at 20 °C, while rarer and more specific taxa became predominant over time, at more extreme temperatures. This suggested that less abundant taxa might be responsible for maintaining the biotransformation capability in the activated sludge at extreme temperatures. Micropollutant biotransformation rates mostly deviated from the classic Arrhenius model below 15 °C and above 25 °C, indicating that prolonged exposure to temperature changes leads to temperature-induced taxonomic shifts, resulting in the emerging of different sets of biotransformation pathways over different temperature ranges.

Isotope signatures of N2O in a mixed microbial population system: constraints on N2O producing pathways in wastewater treatment.

We present measurements of site preference (SP) and bulk (15)N/(14)N ratios (δ(15)N(bulk)(N2O)) of nitrous oxide (N(2)O) by quantum cascade laser absorption spectroscopy (QCLAS) as a powerful tool to investigate N(2)O production pathways in biological wastewater treatment. QCLAS enables high-precision N(2)O isotopomer analysis in real time. This allowed us to trace short-term fluctuations in SP and δ(15)N(bulk)(N2O) and, hence, microbial transformation pathways during individual batch experiments with activated sludge from a pilot-scale facility treating municipal wastewater. On the basis of previous work with microbial pure cultures, we demonstrate that N(2)O emitted during ammonia (NH(4)(+)) oxidation with a SP of -5.8 to 5.6 ‰ derives mostly from nitrite (NO(2)(-)) reduction (e.g., nitrifier denitrification), with a minor contribution from hydroxylamine (NH(2)OH) oxidation at the beginning of the experiments. SP of N(2)O produced under anoxic conditions was always positive (1.2 to 26.1 ‰), and SP values at the high end of this spectrum (24.9 to 26.1 ‰) are indicative of N(2)O reductase activity. The measured δ(15)N(bulk)(N2O) at the initiation of the NH(4)(+) oxidation experiments ranged between -42.3 and -57.6 ‰ (corresponding to a nitrogen isotope effect Δδ(15)N = δ(15)N(substrate) - δ(15)N(bulk)(N2O) of 43.5 to 58.8 ‰), which is considerably higher than under denitrifying conditions (δ(15)N(bulk)(N2O) 2.4 to -17 ‰; Δδ(15)N = 0.1 to 19.5 ‰). During the course of all NH(4)(+) oxidation and nitrate (NO(3)(-)) reduction experiments, δ(15)N(bulk)(N2O) increased significantly, indicating net (15)N enrichment in the dissolved inorganic nitrogen substrates (NH(4)(+), NO(3)(-)) and transfer into the N(2)O pool. The decrease in δ(15)N(bulk)(N2O) during NO(2)(-) and NH(2)OH oxidation experiments is best explained by inverse fractionation during the oxidation of NO(2)(-) to NO(3)(-).

Wastewater microorganisms impact the micropollutant biotransformation potential of natural stream biofilms.

Biotransformation is the most important process removing manmade chemicals from the environment, yet mechanisms governing this essential ecosystem function are underexplored. To understand these mechanisms, we conducted experiments in flow-through systems, by colonizing stream biofilms under different conditions of mixing river water with treated (and ultrafiltered) wastewater. We performed biotransformation experiments with those biofilms, using a set of 75 micropollutants, and could disentangle potential mechanisms determining the biotransformation potential of stream biofilms. We showed that the increased biotransformation potential downstream of wastewater treatment plants that we observed for specific micropollutants contained in household wastewaters (downstream effect) is caused by microorganisms released with the treated effluent, rather than by the in-stream exposure to those micropollutants. Complementary data from 16S rRNA amplicon-sequencing revealed 146 amplicon sequence variants (ASVs) that followed the observed biotransformation patterns. Our results align with findings for community tolerance, and provide clear experimental evidence that microorganisms released with treated wastewater integrate into downstream biofilms and impact crucial ecosystem functions.

Wastewater microorganisms impact microbial diversity and important ecological functions of stream periphyton.

Effluents of wastewater treatment plants can impact microbial communities in the receiving streams. However, little is known about the role of microorganisms in wastewater as opposed to other wastewater constituents, such as nutrients and micropollutants. We aimed therefore at determining the impact of wastewater microorganisms on the microbial diversity and function of periphyton, key microbial communities in streams. We used a flow-through channel system to grow periphyton upon exposure to a mixture of stream water and unfiltered or ultra-filtered wastewater. Impacts were assessed on periphyton biomass, activities and tolerance to micropollutants, as well as on microbial diversity. Our results showed that wastewater microorganisms colonized periphyton and modified its community composition, resulting for instance in an increased abundance of Chloroflexi and a decreased abundance of diatoms and green algae. This led to shifts towards heterotrophy, as suggested by the changes in nutrient stoichiometry and the increased mineralization potential of carbon substrates. An increased tolerance towards micropollutants was only found for periphyton exposed to unfiltered wastewater but not to ultra-filtered wastewater, suggesting that wastewater microorganisms were responsible for this increased tolerance. Overall, our results highlight the need to consider the role of wastewater microorganisms when studying potential impacts of wastewater on the receiving water body.

Tracing N2O formation in full-scale wastewater treatment with natural abundance isotopes indicates control by organic substrate and process settings.

Nitrous oxide (N2O) dominates greenhouse gas emissions in wastewater treatment plants (WWTPs). Formation of N2O occurs during biological nitrogen removal, involves multiple microbial pathways, and is typically very dynamic. Consequently, N2O mitigation strategies require an improved understanding of nitrogen transformation pathways and their modulating controls. Analyses of the nitrogen (N) and oxygen (O) isotopic composition of N2O and its substrates at natural abundance have been shown to provide valuable information on formation and reduction pathways in laboratory settings, but have rarely been applied to full-scale WWTPs. Here we show that N-species isotope ratio measurements at natural abundance level, combined with long-term N2O monitoring, allow identification of the N2O production pathways in a full-scale plug-flow WWTP (Hofen, Switzerland). Heterotrophic denitrification appears as the main N2O production pathway under all tested process conditions (0-2 mgO2/l, high and low loading conditions), while nitrifier denitrification was less important, and more variable. N2O production by hydroxylamine oxidation was not observed. Fractional N2O elimination by reduction to dinitrogen (N2) during anoxic conditions was clearly indicated by a concomitant increase in site preference, δ18O(N2O) and δ15N(N2O). N2O reduction increased with decreasing availability of dissolved inorganic N and organic substrates, which represents the link between diurnal N2O emission dynamics and organic substrate fluctuations. Consequently, dosing ammonium-rich reject water under low-organic-substrate conditions is unfavorable, as it is very likely to cause high net N2O emissions. Our results demonstrate that monitoring of the N2O isotopic composition holds a high potential to disentangle N2O formation mechanisms in engineered systems, such as full-scale WWTP. Our study serves as a starting point for advanced campaigns in the future combining isotopic technologies in WWTP with complementary approaches, such as mathematical modeling of N2O formation or microbial assays to develop efficient N2O mitigation strategies.

Combined nitritation-anammox: advances in understanding process stability.

Efficient nitrogen removal from wastewater containing high concentrations of ammonium but little organic substrate has recently been demonstrated by several full-scale applications of the combined nitritation-anammox process. While the process efficiency is in most cases very good, process instabilities have been observed to result in temporary process failures. In the current study, conditions resulting in instability and strategies to regain efficient operation were evaluated. First, data from full-scale operation is presented, showing a sudden partial loss of activity followed by recovery within less than 1 month. Results from laboratory-scale experiments indicate that these dynamics observed in full scale can be caused by partial inhibition of the ammonia oxidizing bacteria (AOB), while anammox inhibition is a secondary effect due to temporarily reduced O(2) depletion. Complete anammox inhibition is observed at 0.2 mg O(2) · L(-1), resulting in NO(2)(-) accumulation. However, this inhibition of anammox is reversible within minutes after O(2) depletion. Thus, variable AOB activity was identified as the key to reactor stability. With appropriate interpretation of the online NH(4)(+) signal, accumulation of NO(2)(-) can be detected indirectly and used to signal an imbalance of O(2) supply and AOB activity (no suitable online NO(2)(-) electrode is currently available). Second, increased abundance of nitrite-oxidizing bacteria (NOB; competing with anammox for NO(2)(-)) is known as another cause of instability. Based on a comparison of parallel full-scale reactors, it is suggested that an infrequent and short-term increased O(2) supply (e.g., for maintenance of aerators) that exceeds prompt depletion of oxygen by AOB may have caused increased NOB abundance. The volumetric air supply as a proxy for O(2) supply thus needs to be linked to AOB activity. Further, NOB can be washed out of the system during regular operation if the system is operated at a sludge age in the range of 45 days and by controlling the air supply according to the NO(3)(-) concentration in the treated effluent. Early detection of growing NOB abundance while the population is still low can help guide process operation and it is suggested that molecular methods of quantifying NOB abundance should be tested.

N2O emissions: modeling the effect of process configuration and diurnal loading patterns.

The objective of this research was to develop a mechanistic model for quantifying N2O emissions from activated sludge plants and demonstrate how this may be used to evaluate the effects of process configuration and diurnal loading patterns. The model describes the mechanistic link between the factors recognized to correlate positively with N2O emissions. The primary factors are the presence of ammonia and nitrite accumulation. Low dissolved oxygen concentrations also may be implicated through differential impacts on ammonia-oxidizing bacteria (AOB) versus nitrite-oxidizing bacteria (NOB) activity. Factors promoting N2O emissions at treatment plants are discussed below. The model was applied to data from laboratory and pilot-scale systems. From a practical standpoint, plant configuration (e.g., plug-flow versus complete-mix), influent loading patterns (and peak load), and certain operating strategies (e.g., handling of return streams) are all important in determining N2O emissions.

Application of UV absorbance and electron-donating capacity as surrogates for micropollutant abatement during full-scale ozonation of secondary-treated wastewater.

Ozonation of secondary-treated wastewater for the abatement of micropollutants requires a reliable control of ozone doses. Changes in the UV absorbance of dissolved organic matter (DOM) during ozonation allow to estimate micropollutant abatement on-line and were therefore identified as feed-back control parameter. In this study, the suitability of the electron-donating capacity (EDC) as an additional surrogate parameter which is independent of optical DOM properties was evaluated during full-scale ozonation. For this purpose, a recently developed EDC analyzer was enhanced to enable continuous on-line EDC and UV absorbance measurements. During a multi-week monitoring campaign at the wastewater treatment plant of Zurich, Switzerland, specific ozone doses were varied from 0.13 to 0.91 mgO3⋅mgDOC-1 and selected micropollutants with different ozone reactivities were analyzed by LC-MS in conjunction with bromate analysis by IC-MS. In agreement with previous laboratory studies, the relative residual UV absorbance and EDC both decreased exponentially as a function of the specific ozone dose and, in comparison to the residual UV absorbance, residual EDC values showed a more pronounced decrease at low specific ozone doses ≤0.34 mgO3⋅mgDOC-1. Logistic regression models allowed to estimate relative residual micropollutant concentrations in the ozonation effluent using either the residual UV absorbance or EDC as explanatory variable. Averaging those models along the explanatory variables allowed to estimate target values in relative residual UV absorbances and EDC for specific micropollutant abatement targets. In addition, both parameters allowed to identify conditions with elevated conversions of bromide to bromate. Taken together, these findings show that the integration of relative residual EDC values as a second control parameter can improve existing absorbance-based ozonation control systems to meet micropollutant abatement targets, particularly for treatment systems where low ozone doses are applied.

Robustness of mainstream anammox activity at bench and pilot scale.

New technologies and processes, such as mainstream anammox, aim to reduce energy requirements of wastewater treatment and improve effluent quality. However, in municipal wastewater (MWW) anammox system are often unstable due to process control disturbance, influent variability, or unwanted nitrite oxidizing bacteria (NOB). This study examines the anammox system by focusing on anammox activity and its robustness in a mainstream environment. An 8 m3 pilot-scale sequencing batch reactor (SBR) receiving pretreated MWW (with external nitrite addition) was seeded with pre-colonized carriers. Within six months at 12-20 °C an anammox activity of 200 gN·m-3·d-1 was achieved. After the startup an anammox activity of 260 ± 83 gN·m-3·d-1 was maintained over 450 days. The robustness of the anammox activity was analyzed through three disturbance experiments. Anammox biofilm on carriers were exposed to dissolved oxygen (DO = 1.6 mg·L-1, intermittent aeration), organic loading rate (OLR, C/N increased from 2:1 to 5:1) and temperature disturbances (20 °C to 12 °C) in triplicate 12 L bench scale reactors. The anammox activity and microbial community was monitored during these disturbances. The DO and OLR disturbance experiments were replicated at pilot scale to investigate upscaling effects. Bench and pilot scale anammox activity were unaffected by the DO disturbance. Similarly, an increase in OLR did not deteriorate the bench and pilot scale anammox activity, if nitrate was available. When, at bench scale, the reactor temperature was reduced from 20 °C to 12 °C overnight, anammox activity decreased significantly, this was not the case for the slow seasonal temperature changes (12-25 °C) at pilot scale where no strong temperature dependency was detected in winter. Metagenomic analysis revealed a broad range of Brocadiaceae species with no single dominant anammox species. Anammox thrive under mainstream conditions and can withstand typical process disruptions.

Linking seasonal N2O emissions and nitrification failures to microbial dynamics in a SBR wastewater treatment plant.

Nitrous oxide (N2O) is a strong greenhouse gas and causal for stratospheric ozone depletion. During biological nitrogen removal in wastewater treatment plants (WWTP), high N2O fluxes to the atmosphere can occur, typically exhibiting a seasonal emission pattern. Attempts to explain the peak emission phases in winter and spring using physico-chemical process data from WWTP were so far unsuccessful and new approaches are required. The complex and diverse microbial community of activated sludge used in biological treatment systems also exhibit substantial seasonal patterns. However, a potentially causal link between the seasonal patterns of microbial diversity and N2O emissions has not yet been investigated. Here we show that in a full-scale WWTP nitrification failure and N2O peak emissions, bad settleability of the activated sludge and a turbid effluent strongly correlate with a significant reduction in the microbial community diversity and shifts in community composition. During episodes of impaired performance, we observed a significant reduction in abundance for filamentous and nitrite oxidizing bacteria in all affected reactors. In some reactors that did not exhibit nitrification and settling failures, we observed a stable microbial community and no drastic loss of species. Standard engineering approaches to stabilize nitrification, such as increasing the aerobic sludge age and oxygen availability failed to improve the plant performance on this particular WWTP and replacing the activated sludge was the only measure applied by the operators to recover treatment performance in affected reactors. Our results demonstrate that disturbances of the sludge microbiome affect key structural and functional microbial groups, which lead to seasonal N2O emission patterns. To reduce N2O emissions from WWTP, it is therefore crucial to understand the drivers that lead to the microbial population dynamics in the activated sludge.

Temperature modulates stress response in mainstream anammox reactors.

Autotrophic nitrogen removal by anaerobic ammonium oxidizing (anammox) bacteria is an energy-efficient nitrogen removal process in wastewater treatment. However, full-scale deployment under mainstream conditions remains challenging for practitioners due to the high stress susceptibility of anammox bacteria towards fluctuations in dissolved oxygen (DO) and temperature. Here, we investigated the response of microbial biofilms with verified anammox activity to DO shocks under 20 °C and 14 °C. While pulse disturbances of 0.3 mg L-1 DO prompted only moderate declines in the NH4+ removal rates, 1.0 mg L-1 DO led to complete but reversible inhibition of the NH4+ removal activity in all reactors. Genome-centric metagenomics and metatranscriptomics were used to investigate the stress response on various biological levels. We show that temperature regime and strength of DO perturbations induced divergent responses from the process level down to the transcriptional profile of individual taxa. Community-wide gene expression differed significantly depending on the temperature regime in all reactors, and we found a noticeable impact of DO disturbances on genes involved in transcription, translation, replication and posttranslational modification at 20 °C but not 14 °C. Genome-centric analysis revealed that different anammox species and other key biofilm taxa differed in their transcriptional responses to distinct temperature regimes and DO disturbances.

Distinct growth stages controlled by the interplay of deterministic and stochastic processes in functional anammox biofilms.

Mainstream anaerobic ammonium oxidation (anammox) represents one of the most promising energy-efficient mechanisms of fixed nitrogen elimination from wastewaters. However, little is known about the exact processes and drivers of microbial community assembly within the complex microbial biofilms that support anammox in engineered ecosystems. Here, we followed anammox biofilm development on fresh carriers in an established 8m3 mainstream anammox reactor that is exposed to seasonal temperature changes (~25-12°C) and varying NH4+ concentrations (5-25 mg/L). We use fluorescence in situ hybridization and 16S rRNA gene sequencing to show that three distinct stages of biofilm development emerge naturally from microbial community composition and biofilm structure. Neutral modelling and network analysis are employed to elucidate the relative importance of stochastic versus deterministic processes and synergistic and antagonistic interactions in the biofilms during their development. We find that the different phases are characterized by a dynamic succession and an interplay of both stochastic and deterministic processes. The observed growth stages (Colonization, Succession and Maturation) appear to be the prerequisite for the anticipated growth of anammox bacteria and for reaching a biofilm community structure that supports the desired metabolic and functional capacities observed for biofilm carriers already present in the system (~100gNH4-N m3 d-1). We discuss the relevance of this improved understanding of anammox-community ecology and biofilm development in the context of its practical application in the start-up, configuration, and optimization of anammox biofilm reactors.

Nitrogen isotope effects can be used to diagnose N transformations in wastewater anammox systems.

Anaerobic ammonium oxidation (anammox) plays an important role in aquatic systems as a sink of bioavailable nitrogen (N), and in engineered processes by removing ammonium from wastewater. The isotope effects anammox imparts in the N isotope signatures (15N/14N) of ammonium, nitrite, and nitrate can be used to estimate its role in environmental settings, to describe physiological and ecological variations in the anammox process, and possibly to optimize anammox-based wastewater treatment. We measured the stable N-isotope composition of ammonium, nitrite, and nitrate in wastewater cultivations of anammox bacteria. We find that the N isotope enrichment factor 15ε for the reduction of nitrite to N2 is consistent across all experimental conditions (13.5‰ ± 3.7‰), suggesting it reflects the composition of the anammox bacteria community. Values of 15ε for the oxidation of nitrite to nitrate (inverse isotope effect, - 16 to - 43‰) and for the reduction of ammonium to N2 (normal isotope effect, 19-32‰) are more variable, and likely controlled by experimental conditions. We argue that the variations in the isotope effects can be tied to the metabolism and physiology of anammox bacteria, and that the broad range of isotope effects observed for anammox introduces complications for analyzing N-isotope mass balances in natural systems.

Impact of wastewater on the microbial diversity of periphyton and its tolerance to micropollutants in an engineered flow-through channel system.

Wastewater treatment plants (WWTPs) play an important role in retaining organic matter and nutrients but to a lesser extent micropollutants. Therefore, treated wastewater is recognized as a major source of multiple stressors, including complex mixtures of micropollutants. These can potentially affect microbial communities in the receiving water bodies and the ecological functions they provide. In this study, we evaluated in flow-through channels the consequences of an exposure to a mixture of stream water and different percentages of urban WWTP effluent, ranging from 0% to 80%, on the microbial diversity and function of periphyton communities. Assuming that micropollutants exert a selective pressure for tolerant microorganisms within communities, we further examined the periphyton sensitivity to a micropollutant mixture extracted from passive samplers that were immersed in the wastewater effluent. As well, micropollutants in water and in periphyton were comprehensively quantified. Our results show that micropollutants detected in periphyton differed from those found in water, both in term of concentration and composition. Especially photosystem II inhibitors accumulated in periphyton more than other pesticides. Although effects of other substances cannot be excluded, this accumulation may have contributed to the observed higher tolerance of phototrophic communities to micropollutants upon exposure to 30% and 80% of wastewater. On the contrary, no difference in tolerance was observed for heterotrophic communities. Exposure to the gradient of wastewater led to structural differences in both prokaryotic and eukaryotic communities. For instance, the relative abundance of cyanobacteria was higher with increasing percentage of wastewater effluent, whereas the opposite was observed for diatoms. Such results could indicate that differences in community structure do not necessarily lead to higher tolerance. This highlights the need to consider other wastewater constituents such as nutrients and wastewater-derived microorganisms that can modulate community structure and tolerance. By using engineered flow-through channels that mimic to some extent the required field conditions for the development of tolerance in periphyton, our study constitutes a base to investigate the mechanisms underlying the increased tolerance, such as the potential role of microorganisms originating from wastewater effluents, and different treatment options to reduce the micropollutant load in effluents.

Estimation of countrywide N2O emissions from wastewater treatment in Switzerland using long-term monitoring data.

Nitrous oxides (N2O) emissions contribute to climate change and stratospheric ozone depletion. Wastewater treatment is an important, yet likely underestimated, source of N2O emissions, as recent, long-term monitoring campaigns have demonstrated. However, the available data are insufficient to representatively estimate countrywide emission due to the brevity of most monitoring campaigns. This study showed that the emission estimates can be significantly improved using an advanced approach based on multiple continuous, long-term monitoring campaigns. In monitoring studies on 14 full-scale wastewater treatment plants (WWTPs), we found a strong variability in the yearly emission factors (EFs) (0.1 to 8% of the incoming nitrogen load) which exhibited a good correlation with effluent nitrite. But countrywide data on nitrite effluent concentrations is very limited and unavailable for emission estimation in many countries. Hence, we propose a countrywide emission factor calculated from the weighted EFs of three WWTP categories (carbon removal, EF: 0.1-8%, nitrification only: 1.8%, and full nitrogen removal: 0.9%). However, EF of carbon removal WWTPs are still highly uncertain given the expected variability in performance. The newly developed approach allows representative, country-specific estimations of the N2O emissions from WWTP. Applied to Switzerland, the estimations result in an average EF of 0.9 to 3.6% and total emissions of 410 to 1690 tN2O-N/year, which corresponds to 0.3-1.4% of the total greenhouse gas emissions in Switzerland. Our results demonstrate that better data availability and an improved understanding of long-term monitoring campaigns is crucial to improve current emission estimations. Finally, our results confirm several measures to mitigate N2O emissions from wastewater treatment; year-round denitrification, limiting nitrite accumulation, and stringent control of sludge age in carbon removal plants.