The implementation of autotrophic anaerobic ammonium oxidation processes for the removal of nitrogen from municipal wastewater (known as “mainstream anammox”) bears the potential to bring wastewater treatment plants close to energy autarky. The aim of the present work was to assess the long-term stability of partial nitritation/anammox (PN/A) processes operating at low temperatures and their reliability in meeting nitrogen concentrations in the range of typical discharge limits below 2 mgNH4-N·normalL−1 and 10 mgNtot·L−1. Two main 12-L sequencing batch reactors were operated in parallel for PN/A on aerobically pre-treated municipal wastewater (21 ± 5 mgNH4-N·normalL−1 and residual 69 ± 19 mgCODtot·L−1) for more than one year, including over 5 months at 15 °C. The two systems consisted of a moving bed biofilm reactor (MBBR) and a hybrid MBBR (H-MBBR) with flocculent biomass. Operation at limiting oxygen concentrations (0.15–0.18 mgnormalO2·normalL−1) allowed stable suppression of the activity of nitrite-oxidizing bacteria at 15 °C with a production of nitrate over ammonium consumed as low as 16% in the MBBR. Promising nitrogen removal rates of 20–40 mgN·L−1·d−1 were maintained at hydraulic retention times of 14 h. Stable ammonium and total nitrogen removal efficiencies over 90% and 70% respectively were achieved. Both reactors reached average concentrations of total nitrogen below 10 mgN·L−1 in their effluents, even down to 6 mgN·L−1 for the MBBR, with an ammonium concentration of 2 mgN·L−1 (set as operational threshold to stop aeration). Furthermore, the two PN/A systems performed almost identically with respect to the biological removal of organic micropollutants and, importantly, to a similar extent as conventional treatments. A sudden temperature drop to 11 °C resulted in significant suppression of anammox activity, although this was rapidly recovered after the temperature was increased back to 15 °C. Analyses of 16S rRNA gene-targeted amplicon sequencing revealed that the anammox guild of the bacterial communities of the two systems was composed of the genus “Candidatus Brocadia”. The potential of PN/A systems to compete with conventional treatments for biological nutrients removal both in terms of removal rates and overall effluent quality was proven.

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Direct treatment of municipal wastewater (MWW) based on anaerobic ammonium oxidizing (anammox) bacteria holds promise to turn the energy balance of wastewater treatment neutral or even positive. Currently, anammox processes are successfully implemented at full scale for the treatment of high-strength wastewaters, whereas the possibility of their mainstream application still needs to be confirmed. In this study, the growth of anammox organisms on aerobically pre-treated municipal wastewater (MWWpre-treated), amended with nitrite, was proven in three parallel reactors. The reactors were operated at total N concentrations in the range 5–20 mgN∙L−1, as expected for MWW. Anammox activities up to 465 mgN∙L−1∙d−1 were reached at 29 °C, with minimum doubling times of 18 d. Lowering the temperature to 12.5 °C resulted in a marked decrease in activity to 46 mgN∙L−1∙d−1 (79 days doubling time), still in a reasonable range for autotrophic nitrogen removal from MWW. During the experiment, the biomass evolved from a suspended growth inoculum to a hybrid system with suspended flocs and wall-attached biofilm. At the same time, MWWpre-treated had a direct impact on process performance. Changing the influent from synthetic medium to MWWpre-treated resulted in a two-month delay in net anammox growth and a two to three-fold increase in the estimated doubling times of the anammox organisms. Interestingly, anammox remained the primary nitrogen consumption route, and high-throughput 16S rRNA gene-targeted amplicon sequencing analyses revealed that the shift in performance was not associated with a shift in dominant anammox bacteria (“Candidatus Brocadia fulgida”). Furthermore, only limited heterotrophic denitrification was observed in the presence of easily biodegradable organics (acetate, glucose). The observed delays in net anammox growth were thus ascribed to the acclimatization of the initial anammox population or/and the development of a side population beneficial for them. Additionally, by combining microautoradiography and fluorescence in situ hybridization it was confirmed that the anammox organisms involved in the process did not directly incorporate or store the amended acetate and glucose. In conclusion, these investigations strongly support the feasibility of MWW treatment via anammox.

BACKGOUND: Proper treatment technologies are required to address the environmental issues associated with increasing volumes of slurries. Ammonia stripping reduces the nitrogen content of the slurries and allows for its recovery in a valuable form. Herein the influence of pig slurry characteristics on ammonia stripping efficiency and the quality of the recovered ammonia solution were assessed. RESULTS: Substrates characterized by low organic matter content, below 10 g COD L -1 , resulted in ammonia stripping efficiencies greater than 80%. Changing slurry pH to 9.5 significantly improved the process, even though high COD contents kept the efficiencies below 70%. Ammonium sulfate solutions could be concentrated up to nitrogen contents greater than 40 g N L -1 , while maintaining low organic contamination. Introducing a basic trap (pH > 12) before the acid one, allowed for the retention of more than 60% of the stripped organics with less than 3% of the stripped ammonia.CONCLUSIONS: Ammonia stripping coupled with absorption proved to be a suitable technical solution for the recovery and valorization of the nitrogen contained in pig slurries. Clear enhancements in process efficiency were observed in the case of slurries with low organic matter content. The introduction of a basic trap, together with a slight increase in the operational pH level, further increased organics abatement.

Combined partial nitritation-anammox (PN/A) systems are increasingly being employed for sustainable removal of nitrogen from wastewater, but process instabilities present ongoing challenges for practitioners. The goal of this study was to elucidate differences in process stability between PN/A process variations employing two distinct aggregate types: biofilm [in moving bed biofilm reactors (MBBRs)] and suspended growth biomass. Triplicate reactors for each process variation were studied under baseline conditions and in response to a series of transient perturbations. MBBRs displayed elevated NH removal rates relative to those of suspended growth counterparts over six months of unperturbed baseline operation but also exhibited significantly greater variability in performance. Transient perturbations led to strikingly divergent yet reproducible behavior in biofilm versus suspended growth systems. A temperature perturbation prompted a sharp reduction in NH removal rates with no accumulation of NO and rapid recovery in MBBRs, compared to a similar reduction in NH removal rates but a high level of accumulation of NO in suspended growth reactors. Pulse additions of a nitrification inhibitor (allylthiourea) prompted only moderate declines in performance in suspended growth reactors compared to sharp decreases in NH removal rates in MBBRs. Quantitative fluorescence in situ hybridization demonstrated a significant enrichment of anammox in MBBRs compared to suspended growth reactors, and conversely a proportionally higher AOB abundance in suspended growth reactors. Overall, MBBRs displayed significantly increased susceptibility to transient perturbations employed in this study compared to that of suspended growth counterparts (stability parameter), including significantly longer recovery times (resilience). No significant difference in the maximal impact of perturbations (resistance) was apparent. Taken together, our results suggest that aggregate architecture (biofilm vs suspended growth) in PN/A processes exerts an unexpectedly strong influence on process stability.

Anammox bacteria enable an efficient removal of nitrogen from sewage in processes involving partial nitritation and anammox (PN/A) or nitrification, partial denitrification, and anammox (N-PdN/A). In mild climates, anammox bacteria must be adapted to 15 C, typically by gradual temperature decrease; however, this takes months or years. To reduce the time necessary for the adaptation, an unconventional method of cold shocks is promising, involving hours-long exposure of anammox biomass to extremely low temperatures. We compared the efficacies of gradual temperature decrease and cold shocks to increase the metabolic activity of anammox (fed-batch reactor, planktonic Ca. Kuenenia). We assessed the cold shock mechanism on the level of protein expression (quantitative shot-gun proteomics, LC-HRMS/MS) and structure of membrane lipids (UPLC-HRMS/MS). The shocked culture was more active (0.66+-0.06 vs 0.48+-0.06 kg-N/kg-VSS/d) and maintained the relative content of N-respiration proteins at levels consistent levels with the initial state, whereas the content of these proteins decreased in gradually acclimated culture. Cold shocks also induced a more efficient up-regulation of cold shock proteins (e.g. CspB, TypA, ppiD). Ladderane lipids characteristic for anammox evolved to a similar end-point in both cultures which confirms their role in anammox bacteria adaptation to cold and indicates a three-pronged adaptation mechanism involving ladderane lipids (ladderane alkyl length, introduction of shorter non-ladderane alkyls, polar headgroup). Overall, we show the outstanding potential of cold shocks for low-temperature adaptation of anammox bacteria and provide yet unreported detailed mechanisms of anammox adaptation to low temperatures.