Evidence of differential adaptation to decreased temperature by anammox bacteria.

Low temperature is recognized as one of the major barriers for the application of the anaerobic ammonium oxidation (anammox) process to treat mainstream wastewater. Studies are yet to reveal the underlying biological limitations and molecular mechanisms associated with the inhibition of low temperature on the anammox process. In this study, metaproteomics was used to examine proteome modulation patterns of the anammox community occurring at different temperatures. The anammox community remarkably altered their proteomes when the temperature decreased from 35 °C to 20 °C. This was especially for proteins involved in energy conversion, transcription and translation and inorganic ion transport. However, at 15 °C the anammox activities became distinctly inhibited, and there was evidence of energy limitations and severe stress in Candidatus Kuenenia and to a lesser degree in Candidatus Brocadia. Candidatus Jettenia exhibited more changes in its proteome at 15 °C. From the proteomes, at the lower temperatures there was evidence of stress caused by toxic nitrogen compounds or reactive oxygen species in the anammox bacteria. Hydroxylamine oxidoreductase (HAO)-like proteins and an oxidative stress response protein (a catalase) were in high abundance to potentially ameliorate these inhibitory effects. This study offers metaproteomic insight into the anammox community-based physiological response to decreasing temperatures.

Expression of the nirS, hzsA, and hdh Genes in Response to Nitrite Shock and Recovery in Candidatus Kuenenia stuttgartiensis.

In this study, Candidatus Kuenenia stuttgartiensis were subjected to distinct nitrite shocks (66 (control), 200, 300, 400, and 500 mg N/L), and the responses of mRNA levels of cytochrome cd1 nitrite/nitric oxide oxidoreductase (nirS), hydrazine synthase (hzsA), and hydrazine dehydrogenase (hdh) were assessed. Changes in the hydrazine dehydrogenase (HDH) protein level were monitored. At 200 mg NO2(-)-N/L, the normalized specific anaerobic ammonium-oxidizing activity (nSAA) slightly increased relative to the control despite a significant decrease in nirS, hzsA, and hdh mRNA levels. When nitrite increased to 300 and 400 mg N/L, increased nirS, hzsA, and hdh mRNA levels were observed, but the nSAA decreased, relative to the 200 mg NO2(-)-N/L exposure. HDH protein detection revealed that Candidatus Kuenenia stuttgartiensis attempted to yield high enzyme levels by stimulating mRNA synthesis to resist the nitrite-induced stress. On 500 mg NO2(-)-N/L shock, the nirS, hzsA, and hdh mRNA levels decreased, alongside decreased nSAA and HDH levels. Although the mRNA levels did not always coincide with activities, our findings advance understanding of the mechanisms that anammox bacteria use to cope with nitrite inhibition at the transcriptional and translational levels, which will improve the diagnostic accuracy of bioreactor failures when nitrite accumulation occurs.

Bottom-Up Construction of Mesoporous Cerium-Doped Titania with Stably Dispersed Pt Nanocluster for Efficient Hydrogen Evolution.

Hydrogen generation is one of the crucial technologies to realize sustainable energy development, and the design of advanced catalysts with efficient interfacial sites and fast mass transfer is significant for hydrogen evolution. Herein, an in situ coassembly strategy was proposed to engineer a cerium-doped ordered mesoporous titanium oxide (mpCe/TiO2), of which the abundant oxygen vacancies (Ov) and highly exposed active pore walls contribute to good stability of ultrasmall Pt nanoclusters (NCs, ∼ 1.0 nm in diameter) anchored in the uniform mesopores (ca. 20 nm). Consequently, the tailored mpCe/TiO2 with 0.5 mol % Ce-doping-supported Pt NCs (Pt-mpCe/TiO2-0.5) exhibits superior H2 evolution performance toward the water-gas shift reaction with a 0.73 molH2·s-1·molPt-1 H2 evolution rate at 200 °C, which is almost 6-fold higher than the Pt-mpTiO2 (0.13 molH2·s-1·molPt-1 H2). Density functional theory calculations confirm that the structure of Ce-doped TiO2 with Ce coordinated to six O atoms by substituting Ti atoms is thermodynamically favorable without the deformation of Ti-O bonds. The Ov generated by the six O atom-coordinated Ce doping is highly active for H2O dissociation with an energy barrier of 2.18 eV, which is obviously lower than the 2.37 eV for the control TiO2. In comparison with TiO2, the resultant Ce/TiO2 support acts as a superior electron acceptor for Pt NCs and causes electron deficiency at the Pt/support interface with a 0.17 eV downshift of the Pt d-band center, showing extremely obvious electronic metal-support interaction (EMSI). As a result, abundant and hyperactive Ti3+-Ov(-Ce3+)-Ptδ+ interfacial sites are formed to significantly promote the generation of CO2 and H2 evolution. In addition, the stronger EMSI between Pt NCs and mpCe/TiO2-0.5 than that between Pt and mpTiO2 contributes to the superior self-enhanced catalytic performance during the cyclic test, where the CO conversion at 200 °C increases from 72% for the fresh catalyst to 99% for the used one. These findings reveal the subtle relationship between the mesoporous metal oxide-metal composite catalysts with unique chemical microenvironments and their catalytic performance, which is expected to inspire the design of efficient heterogeneous catalysts.

A novel anammox granules-circulating expanded granular sludge bed reactor for the efficient circulation and retention of floating anammox granules.

In application of anammox process, the operation of the conventional expanded granular sludge bed reactor (EGSBconv.) is severely limited by the blocking and decay of floating anammox granules.To address this emerging issue, a novel three-phase separator configuration was designed and an anammox granules circulating EGSB (EGSBGC) was proposed in this study. In the EGSBGC, an influent scour on floating granules, whose effect was confirmed by simulation with a three-dimensional flow model, was obtained by integrating the external three-phase separator with the influent and the external cycle. After 166-d operation, the nitrogen removal efficiency of the EGSBGC reached 75.6%, being 1.28-times that of the EGSBconv. (58.9%). The sludge concentration in the main body of the EGSBGC reached 3112 ±â€¯65 mg/L, compared with 2613 ±â€¯42 mg/L in the EGSBconv. (p < 0.05). Moreover, the severe granules blockage and decay problem that is frequently encountered in the EGSBconv. no longer occurred in the EGSBGC. The relative abundance of anammox bacteria in granules from the three-phase separator of the EGSBGC was 29.7%, significantly higher than that from the EGSBconv. (16.1%, p < 0.05). The blockage and decay of granules in the three-phase separator of the EGSBconv. led to an obvious proliferation of heterotrophic bacteria, with their relative abundance increased by 9.4% compared with the seed sludge (38.6% vs. 29.2%). This study proposed a practical three-phase separator configuration to sustain efficient and stable operation of anammox processes toward the promotion of granules circulation, retention and reaction.

Enhancement of the adaptability of anammox granules to zinc shock by appropriate organic carbon treatment.

Heavy metals, which are commonly present in high ammonia-containing wastewater, can cause inhibitory effects to anammox reaction. This study proposes a novel approach to enhance the adaptability of anammox granules to heavy metal [Zn(II)] shock by organic carbon (sodium acetate, NaAc) treatment, paying special attention to optimization of the treatment dosage and duration. For granules treated with 200 mg chemical oxygen demand (COD)/L NaAc for 2 d, the activity recovery (six cycles) efficiency after Zn(II) (40 mg/L) shock reached 127.4%. The extracellular polymeric substance (EPS) production increased by 168% and heterotrophic bacteria mildly proliferated (increased by 14%) in such granules compared with the control. The dramatic recovery capacity was likely due to the entrapment and barrier function of EPS and the outer-layer proliferated heterotrophic bacteria. This finding offers a useful process to enable maximum adaptability of anammox granules from heavy metals shocks, allowing anammox technology to be more widely applied.

Microstructure of anammox granules and mechanisms endowing their intensity revealed by microscopic inspection and rheometry.

The anammox process represents a sustainable and cost-effective technique for nitrogen removal from wastewater, where granulation of anammox bacteria could be of great benefit to the system performance. However, knowledge of the specific properties of anammox granules is currently unsatisfactory. In this study, the organization of anammox granules was comprehensively studied from macro to micro scale with a range of microscale techniques. Scanning and transmission electron microscopy and multiple fluorescence labeling combined with confocal laser scanning microscopy were included. Simultaneously, the associated mechanical properties were studied in-depth by rheometry in combination with selective enzymatic hydrolysis. Anammox granules follow a tertiary organization regime, where interactions between individual anammox bacteria made up the primary base, then, the grouping of anammox bacterial cells encapsulated within a thin extracellular polymeric substance (EPS) layer comprised a second arrangement level, and, finally, the cementing of these groups together with other bacteria and polymers gave rise to compact aggregates. α-Polysaccharides and proteins were considered the backbones of anammox granules, contributing greatly to their excellent intensity. ß-Polysaccharides concentrated at the outer rims of anammox granules and combined with other macromolecules to form a buffer zone or protective barrier, beneath which anammox bacteria proliferated. Divalent cationic bridging for EPS binding was prevalent and of great significance within the dense anammox granules, while there was also much weak monovalent ionic interaction. The specific organization and composition of anammox granules endows them with excellent intensity and integrity, which can be of importance for full-scale reactor operations where diverse shocks can be expected.

Comparison of endogenous metabolism during long-term anaerobic starvation of nitrite/nitrate cultivated denitrifying phosphorus removal sludges.

Denitrifying phosphorus removal (DPR) by denitrifying phosphorus-accumulating organisms (DPAOs) is a promising approach for reducing energy and carbon usage. However, influent fluctuations or interruptions frequently expose the DPAOs biomass to starvation conditions, reducing biomass activity and amount, and ultimately degrading the performance of DPR. Therefore, a better understanding of the endogenous metabolism and recovery ability of DPAOs is urgently required. In the present study, anaerobic starvation (12 days) and recovery were investigated in nitrite- and nitrate-cultivated DPAOs at 20 ± 1 °C. The cell decay rates in nitrite-DPAOs sludges from the end of the anaerobic and aerobic phase were 0.008 day⁻¹ and 0.007 day⁻¹, respectively, being 64% and 68% lower than those of nitrate-DPAOs sludges. Nitrite-DPAOs sludges also recovered more rapidly than nitrate-DPAOs sludge after 12 days of starvation. The maintenance energy of nitrite-DPAOs sludges from the end of the anaerobic and aerobic phase were approximately 31% and 34% lower, respectively, than those of nitrate-DPAOs sludges. Glycogen and polyphosphate (poly-P) sequentially served as the main maintenance energy sources in both nitrite-and nitrate-DPAOs sludges. However, the transformation pathway of the intracellular polymers during starvation differed between them. Nitrate-DPAOs sludge used extracellular polymeric substances (EPS) (mainly polysaccharides) as an additional maintenance energy source during the first 3 days of starvation. During this phase, EPS appeared to contribute to 19-27% of the ATP production in nitrate-DPAOs, but considerably less to the cell maintenance of nitrite-DPAOs. The high resistance of nitrite-DPAOs to starvation might be attributable to frequent short-term starvation and exposure to toxic substances such as nitrite/free nitrous acids in the parent nitrite-fed reactor. The strong resistance of nitrite-DPAOs sludge to anaerobic starvation may be exploited in P removal by shortcut denitrification processes.

Identification of the function of extracellular polymeric substances (EPS) in denitrifying phosphorus removal sludge in the presence of copper ion.

Industrial wastewater containing heavy metals that enters municipal wastewater treatment plants inevitably has a toxic impact on biological treatment processes. In this study, the impact of Cu(II) (0, 1.5, 2, 2.5, 3 mg/L) on the performance of denitrifying phosphorus removal (DPR) and microbial community structures was investigated. Particularly, the dynamic change in the amount and composition of extracellular polymeric substances (EPS), and the role of EPS in P removal, were assessed using three-dimensional excitation-emission matrix fluorescence spectroscopy combined with parallel factor (PARAFAC) analysis. The results showed that, after long-term adjustment, the P removal efficiency was maintained at 95 ± 2.7% at Cu(II) addition up to 2.5 mg/L, but deteriorated when the Cu(II) addition was 3 mg/L. The EPS content, including proteins and humic substances, increased with increasing Cu(II) additions at concentrations ≤2.5 mg/L. This property of EPS was beneficial for protecting phosphate-accumulating organisms (PAOs) against heavy metals, as both proteins and humic substances are strong ligands for Cu(II). Therefore, the PAOs abundance was still relatively high (67 ± 3%) when Cu(II) accumulation in sludge was up to 10 mg/g SS. PARAFAC confirmed that aromatic proteins could be transformed into soluble microbial byproduct-like material when microorganisms were subjected to Cu(II) stress, owing to their strong metal ion complexing capacity. The increase in the percentage of humic-like substances enhanced the detoxification function of the sludge EPS. EPS accounted for approximately 26-47% of P removed by adsorption when Cu(II) additions were between 0 and 2.5 mg/L. The EPS function, including binding toxic heavy metals and P storage, enhanced the operating stability of DPR systems. This study provides us with a better understanding of (1) the tolerance of DPR sludge to copper toxicity and (2) the function of sludge EPS in the presence of heavy metals in biological P removal systems.