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.

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.

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.