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Anammox plays a pivotal role in both natural and engineered systems as a process that simultaneously converts fixed nitrogen to N2 and regenerates NO3–. In aquatic and terrestrial ecosystems, isotopic measurements, especially of the NO3– pool, provide an essential constraint on the processes that regulate the supply and elimination of fixed nitrogen, but the isotope effects of anammox remain poorly constrained.We present measurements of the δ15N and δ18O of NO3–, NO2–, and NH4+ as processed by anammox in a mixed microbial community enriched for N removal from wastewater. We find that oxygen isotope effects expressed in NO2– include a substantial contribution from equilibration reactions with water superimposed on kinetic isotope effects. Equilibrium between water and NO2– during processing by anammoxis greatly accelerated above rates observed under abiotic conditions even during growth phases when NO2– is rapidly being consumed. In turn, δ18O of NO3– nearly completely reflects the incorporation of O atoms derived from water with little additional isotopic fractionation. The δ15N values of NO3– and NO2– also show evidence for an equilibrium isotope exchange reaction between these molecules, which raises the possibility that nitrite oxidation is partially reversible, while introducing a high degree of variability into the δ15N of NO3– generated by anammox. Finally, variation observed in the δ15N of NH4+ consumed by anammox can be connected to physiological limitations within the anammox cell.Despite this complexity, we were able to use NO2– and NO3– isotope measurements to diagnose changes in the activity of anammox and related processes within the wastewater treatment system during a low-temperature perturbation experiment. These results provide new constraints for interpreting the variability in δ15N and δ18O of NO3–in natural systems, with implications for estimating relative rates of fixed N turnover processes.

Natural and engineered nitrogen (N) removal processes in aqueous systems represent important sources of nitrogenous gas emissions, including the potent greenhouse gas nitrous dioxide (N2O). The relevance of microbial and abiotic formation pathways can be assessed using 15N tracing techniques. While 15N-N2O analysis using optical analyzers is straightforward, quantification of 15N fractions in inorganic N compounds, ammonium (NH4+), nitrite (NO2-), and nitrate (NO3-), is typically time-consuming and labor-intensive.In this study, we developed an automated sample-preparation unit coupled to a membrane-inlet quadrupole mass spectrometer (3n-ASSP-MIMS) for the online quasi-simultaneous analysis of 15N fractions in NH4+, NO2-, and NO3-. The technique was designed and validated for applications at moderate (100 - 200 μmol L-1) to high (2 – 3 mmol L-1) N, as found in sewer systems, wastewater in treatment plants, or eutrophic surface waters, and 15N spiking (f15) between 1 and 33%.The potential of 3n-ASSP-MIMS was demonstrated in a feasibility study, where the technique, in conjunction with 15N-N2O analyses by FTIR spectroscopy, was applied to pinpoint nitrifier denitrification as the primary N2O formation pathway during partial NH4+ oxidation to NO2- in a lab-scale sequencing batch reactor.

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., Water Research, 200, ISSN:0043-1354, ISSN:1879-2448

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., Communications Biology, 4, ISSN:2399-3642

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., Scientific Reports, 11 (1), ISSN:2045-2322

Complex microbial biofilms orchestrating mainstream anaerobic ammonium oxidation (anammox) represent one of the most promising energy-efficient mechanisms of fixed nitrogen elimination from anthropogenic waste waters. However, little is known about the ecological processes that are driving microbial community assembly leading to functional anammox biofilms in engineered ecosystems. Here, we use fluorescence in situ hybridization and 16S rRNA sequencing combined with network modelling to elucidate the contribution of stochastic and deterministic processes during anammox biofilm development from first colonization to maturation in a carrier-based anammox reactor. We find that distinct stages of biofilm development emerge naturally in terms of structure and community composition. These stages are characterized by dynamic succession and an interplay of stochastic and deterministic processes. The staged process of biofilm establishment appears to be the prerequisite for the anticipated growth of anammox bacteria and for reaching a biofilm community structure with the desired metabolic capacities. We discuss the relevance of this improved understanding of anammox community ecology and biofilm development concerning its practical application in the start-up and configuration of anammox biofilm reactors.

Stable isotope measurements of nitrogen and oxygen in nitrogen-containing molecules provide important constraints on the sources, sinks and pools of these molecules in the environment. Anammox is one of two known biological processes for converting fixed nitrogen to N2, and through its consumption of ammonium and nitrite and production of nitrate, it impacts the supply of a wide variety of fixed N molecules. Nevertheless, the isotope fractionations associated with the various anammox-associated redox reactions remain poorly constrained. We have measured the isotope effects of anammox in microbial communities enriched for the purpose of nitrogen removal from wastewater by anammox. In this system, we can replicate the ecological complexity exhibited in environmental settings, while also performing controlled experiments. We find that under a variety of conditions, the nitrogen isotope effect for the anaerobic oxidation of ammonium in this system (NH4+ to N2) is between 19‰ and 32‰, that for the reduction of nitrite (NO2– to N2) is between 7‰ and 18‰, and that for the production of nitrate (NO2– to NO3–) is between -16‰ and -43‰. We propose that these ranges reflect both (1) a mixture of signals from different anammox-performing species and (2) variation of the isotope effect associated with the anammox process within a given microbial community under different conditions. We seek to understand further what factors control this variability to better interpret stable isotope measurements of N-bearing molecules in environmental settings.

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., Science of The Total Environment, 796, ISSN:0048-9697, ISSN:1879-1026

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 and temperature. Here, we investigated the response of microbial biofilms with verified anammox activity to oxygen shocks under favorable and cold temperature regimes. Genome-centric metagenomics and metatranscriptomics were used to investigate the stress response on various biological levels. We show that temperature regime and strength of oxygen perturbations induced divergent responses from the process level down to the transcriptional profile of individual taxa. Temperature induced distinct transcriptional states in compositionally identical communities and transient pulses of dissolved oxygen resulted in the upregulation of stress-response only under favorable temperatures. Anammox species and other key biofilm taxa display different transcriptional responses to the induced stress regimes.