External redox couple enhanced anammox sludge activity at low temperature: Insight into intracellular resource synthesis.

Anaerobic ammonium oxidation (anammox), an energy-efficient deamination biotechnology, faces operational challenges in low-temperature environments. Enhancing the metabolic activity of anammox bacteria (AnAOB) is pivotal for advancing its application in mainstream municipal wastewater treatment. Inspired by the metabolic adaptability of AnAOB and based on our previous findings, this work investigated the enhancement of intracellular ATP and NADH synthesis through the exogenous supply of reduced humic acid (HAred) and H2O2 redox couple, aiming to augment AnAOB activity under low-temperature conditions. Our experimental setup involved continuous dosing of 0.0067 µmol g-1 volatile suspended solid of H2O2 and 10 mg g-1 volatile suspended solid of HAred into a mainstream anammox reactor operated at 15 °C with an influent TN content of 60 mg/L. The results showed that HAred / H2O2 couple succeeded in maintaining the effluent TN at 10.72 ± 0.91 mg l-1. The specific anammox activity, ATP and NADH synthesis levels of sludge increased by 1.34, 2.33 and 6.50 folds, respectively, over the control setup devoid of the redox couple. High-throughput sequencing analysis revealed that the relative abundance of Candidatus Kuenenia after adding HAred / H2O2 couple reached 3.65 % at the end of operation, which was 5.14 folds higher than that of the control group. Further metabolomics analysis underscored an activation in the metabolism of amino acids, nucleotides, and phospholipids, which collectively enhanced the availability of ATP and NADH for the respiratory processes. These findings may provide guidance on strategy development for improving the electron transfer efficiency of AnAOB and underscore the potential of using redox couples to promote the mainstream application of anammox technology.

Light-Driven Electron Uptake from Nonfermentative Organic Matter to Expedite Nitrogen Dissimilation by Chemolithotrophic Anammox Consortia.

Nonphotosynthetic microorganisms are typically unable to directly utilize light energy, but light might change the metabolic pathway of these bacteria indirectly by forming intermediates such as reactive oxygen species (ROS). This work investigated the role of light on nitrogen conversion by anaerobic ammonium oxidation (anammox) consortia. The results showed that high intensity light (>20000 lx) caused ca. 50% inhibition of anammox activity, and total ROS reached 167% at 60,000 lx. Surprisingly, 200 lx light was found to induce unexpected promotion of the nitrogen conversion rate, and ultraviolet light (<420 nm) was identified as the main contributor. Metagenomic and metatranscriptomic analyses revealed that the gene encoding cytochrome c peroxidase was highly expressed only under 200 lx light. 15N isotope tracing, gene abundance quantification, and external H2O2 addition experiments showed that photoinduced trace H2O2 triggered cytochrome c peroxidase expression to take up electrons from extracellular nonfermentative organics to synthesize NADH and ATP, thereby expediting nitrogen dissimulation of anammox consortia. External supplying reduced humic acid into a low-intensity light exposure system would result in a maximal 1.7-fold increase in the nitrogen conversion rate. These interesting findings may provide insight into the niche differentiation and widespread nature of anammox bacteria in natural ecotopes.

A review of anammox-based nitrogen removal technology: From microbial diversity to engineering applications.

The anaerobic ammonium oxidation (anammox) process has the advantages of high efficiency and low energy consumption, so it has broad application prospects in biological denitrification of wastewater. However, the application of anammox technology to existing wastewater treatment is still challenging. The main problems are the insufficient supply of nitrite and the susceptibility of anammox bacteria to environmental factors. In this paper, from the perspective of the diversity of anammox bacteria, the habitats and characteristics of anammox bacteria of different genera were compared. At the same time, laboratory research and engineering applications of anammox technology in treating wastewater from different sources were reviewed, and the progress of and obstacles to the practical application of anammox technology were clarified. Finally, a focus for future research was proposed to intensively study the water quality barrier factors of anammox and its regulation strategies. Meanwhile, a combined process was developed and optimized on this basis.

Inorganic quantum dots - anammox consortia hybrid for stable nitrogen elimination under high-intensity solar-simulated irradiation.

External stimulus such as light irradiation is able to deteriorate intracellular redox homeostasis and induce photooxidative damage to non-photogenic bacteria. Exploiting effective strategies to help bacteria resisting infaust stress is meaningful for achieving a stable operation of biological treatment system. In this work, selenium-doped carbon quantum dots (Se-CQDs) were blended into anaerobic ammonia oxidation (anammox) bacteria and an inorganic nanoparticle-microbe hybrid was successfully fabricated to evaluate its nitrogen removal performance under solar-simulated irradiation. It was found that the specific anammox activity decreased by 29.7 ± 5.2% and reactive oxygen species (ROS) content increased by 134.8 ± 4.1% under 50,000 lux light. Sludge activity could be completely recovered under the optimum dosage of 0.42 mL·(g volatile suspended solid) -1 Se-CQDs. Hydroxyl radical (·OH) and superoxide anion radical (·O2-) were identified as the leading ROS inducing lipid peroxidation and antioxidase function detriment. Also, the structure of ladderane lipids located on anammoxosome was destroyed by ROS and functional genes abundances declined accordingly. Although cell surface coated Se-CQDs could absorb ultraviolet light and partially mitigated the photoinhibition, the direct scavenging of ROS by intracellular Se-CQDs primarily contributed to the cellular redox homeostasis, antioxidase activity recovery and sludge activity improvement. The findings of this work provide in-depth understanding the metabolic response mechanism of anammox consortia to light irradiation and might be valuable for a more stable and sustainable nitrogen removal technology, i.e., algal-bacterial symbiotic system, development.

Response of the mainstream anammox process to the biodegradable carbon sources in the granule-based systems: The difference in self-stratification of the microbial community.

The inevitable introduction of biodegradable carbon sources (such as monosaccharides and volatile fatty acids) originating from pretreatment units might affect the performance of the mainstream anaerobic ammonium oxidation (anammox) process. Two model carbon sources (glucose and acetate) were selected to investigate their effects on granule-based anammox systems under mainstream conditions (70 mg total nitrogen (TN) L-1, 15 °C). At a nitrogen loading rate of 2.87 ± 0.80 kg N m-3 d-1, a satisfactory effluent quality (TN < 10 mg L-1) was achieved in the presence of glucose or acetate at a chemical oxygen demand (COD/N) ratio of 0.5. The contribution of anammox to nitrogen removal decreased with increasing COD/N ratio to 1.0 because the expression of anammox functional genes was inhibited, whereas the expression of denitrifying functional genes was promoted. However, the nitrogen removal efficiency of the two considered reactors was maintained above 80 %. Self-stratification of the microbial community along the reactor height facilitated a functional balance through the retention of anammox bacteria in granules but resulted in washout of denitrifying bacteria in flocs under a high-flow pattern. These findings highlighted the advantages of granule-based systems in the mainstream anammox process due to their inherent biomass self-segregation property and the need for the development of targeted biomass retention strategies.

The response of anaerobic ammonium oxidation process to bisphenol-A: Linking reactor performance to microbial community and functional gene.

As a typical endocrine disruptor, bisphenol A (BPA) has been widely detected in various water bodies. Although the influence of BPA on traditional biological treatment system has been investigated, it is not clear whether it has potential impact on anaerobic ammonium oxidation (anammox) process. The short- and long-term influences of BPA on reactor operational performance, sludge characteristics and microbial community were investigated in this study. Results revealed that 1 and 3 mg L-1 BPA exhibited a limited adverse impact on granular sludge reactor performance. However, exposure of sludge under 10 mg L-1 BPA would cause an obvious inhibition on nitrogen removal rate from 10.3 ± 0.2 to 7.6 ± 0.4 kg N m-3 d-1. BPA would affect granular sludge metabolic substance excretion and lead to effluent dissolved organic content increase. Both the microbial community and redundancy analysis showed that BPA exhibited a negative influence on Ca. Kuenenia but a positive correlation with SBR1031. Low BPA concentration appeared a limited impact on functional genes while 10 mg L-1 BPA would cause decline of hzsA and hdh abundances. The results of this work might be valuable for in-depth understanding the potential influence of endocrine disruptor on anammox sludge.

Molecular spectroscopy and docking simulation revealed the binding mechanism of phenol onto anammox sludge extracellular polymeric substances.

The rapid development of chemical industry has induced to the large amount of phenolic wastewater production. When the promising anaerobic ammonium oxidation (anammox) was employed to treat the industrial wastewater, phenolic compounds would possibly inhibit the microbial performance. Extracellular polymeric substances (EPSs) play an essential role in protecting cells from being intoxicated by phenolic compound while the distinct mechanism remains elusive. In this work, the interaction of phenol with anammox sludge EPSs and transmembrane ammonium transport (Amt) domain was explored at molecular level by using spectral method and molecular docking simulation. It was found that phenol statically quenched the fluorescent components of EPSs and the protein component dominated the interaction between EPSs and phenol. The overall interaction was an entropy-driven process with hydrophobic interaction as the main driving force, and the CO vibration responded preferentially. As phenol continued to penetrate into the cell surface, there were hydrogen bond, hydrophobic interaction force and π-π base-stacking forces between the Amt domain and phenol. The interaction between phenol and amino acid residues of the Amt domain would interfere the NH4+ transport and further affect the activity of anammox sludge. This work is beneficial for in-depth understanding the role of EPSs in protecting anammox sludge from inhibiting by phenolic pollutants.

A review of heavy metals inhibitory effects in the process of anaerobic ammonium oxidation.

Anaerobic ammonium oxidation (anammox) is a promising biological technology for treating ammonium-rich wastewaters. However, due to the high sensitivity of anammox bacteria, many external factors have inhibitory effects on this process. As one of the commonly found toxic substances in wastewater, heavy metals (HMs) are possible to cause inhibition on anammox sludge, which then results in a declined treatment performance. Getting insights into the response mechanism of anammox sludge to HMs is meaningful for its application in treating this kind of wastewater. This review summarized the effect of different HMs on treatment performance of anammox bioreactor. In addition, the mechanism of toxication raised by HMs was discussed. Also, the potential mitigation strategies were summarized and the future prospects were outlooked. This review might provide useful information for both scientific research on and engineering application of anammox process for treating HMs containing wastewater.

Coagulants put phosphate-accumulating organisms at a competitive disadvantage with glycogen-accumulating organisms in enhanced biological phosphorus removal system.

Enhanced biological phosphorus removal (EBPR) process is susceptible to the changed operation condition, which results in an unstable treatment performance. In this work, long-term effect of coagulants addition, aluminum salt for the reactor R1 and iron salt for the reactor R2, on EBPR systems was comprehensively evaluated. Results showed that during the initial 30 days' coagulant addition, effluent chemical oxygen demand and phosphorus can be reduced below 25 and 0.5 mg·L-1, respectively. Further supply of metal salts would stimulate microbial extracellular polymeric substance excretion and induce reactive oxygen species accumulation, which destroyed the cell membrane integrity and deteriorated the phosphorus removal performance. Moreover, coagulants would decrease the relative abundance of Candidatus Accumulibacter while increase the relative abundance of Candidatus Competibacter, leading phosphors accumulating organisms in a disadvantage position. The results of this work might be valuable for the operation of chemical assisted biological phosphorus removal bioreactor.

A review on characterizing the metabolite property of anammox sludge by spectroscopy.

As one of the most promising autotrophic biological nitrogen removal technology, anaerobic ammonia oxidation (anammox) has gained intense attention for the past decades and several full-scale facilities have been implemented worldwide. However, anammox bacteria are easily affected by disturbed external environmental factors, which commonly leads to the fluctuations in reactor performance. The response of anammox sludge to external stress results in changes in components and structural characteristics of intracellular and extracellular polymer substances. Real-time and convenient spectral analysis of anammox sludge metabolites can give early warning of performance deterioration under external stresses, which is of great significance to the stable operation of bioreactor. This review summarized the research progress on characterizing the intracellular and extracellular metabolites of anammox sludge through spectroscopic techniques. The correlation between anammox sludge activity and its key metabolites was analyzed. Also, the limitations and future prospects of applying spectral analytical techniques for anammox bioreactor monitoring were discussed and outlooked. This review may provide valuable information for both scientific study and engineering application of anammox based nitrogen removal technology.

Surface oxygen vacancies formation on Zn2SnO4 for bisphenol-A degradation under visible light: The tuning effect by peroxymonosulfate.

Visible light catalysis has been widely coupled with persulfate activation for refractory pollutants removal, while the exact role of persulfate played in such composite system is still questionable. In this work, the relation between peroxymonosulfate (PMS) induced structure change and visible light responsive activity of inverse spinel: i.e., Zn2SnO4, was deciphered. Under the visible light illumination (λ> 420nm) PMS addition would endow the composite system with pollutant removal performance. Batch test revealed that 60% of bisphenol-A (5 mg L-1) was mineralized within 3 h reaction time, by dosing 0.81 mM PMS and 0.1 g L-1 catalyst. The above oxidative system was also effective for other refractory pollutants elimination. Further analysis indicated that PMS could reduce the band gap of spinel from 2.75 to 2.52 eV and thereby enabling its visible light activity. Photogenerated h+ induced •OH and e- mediated •O2- contributed to the pollutant removal while h+ played a leading role. Density functional theory revealed that PMS would capture oxygen atom of spinel and induce surface oxygen vacancy defect structure formation. Also, three-oxygen atom coordinated Zn was identified as the possible catalyze site. This work is valuable for deep understanding the exact role of persulfate in photocatalytic system.

Molecular Insight into the Binding Property and Mechanism of Sulfamethoxazole to Extracellular Proteins of Anammox Sludge.

Antibiotics are widely found in nitrogen-containing wastewater, which may affect the operation stability of anaerobic ammonium oxidation (anammox)-based biological treatment systems. Extracellular polymeric substances (EPSs) of anammox sludge play a pivotal role in combining with antibiotics; however, the exact role and how the structure of the leading component of EPSs (i.e., extracellular proteins) changes under antibiotic stress remain to be elucidated. Here, the interaction between sulfamethoxazole and the extracellular proteins of anammox sludge was investigated via multiple spectra and molecular simulation. Results showed that sulfamethoxazole statically quenched the fluorescent components of EPSs, and the quenching constant of the aromatic proteins was the largest, with a value of 1.73 × 104 M-1. The overall binding was an enthalpy-driven process, with ΔH = -75.15 kJ mol-1, ΔS = -0.175 kJ mol-1 K-1, and ΔG = -21.10 kJ mol-1 at 35 °C. The O-P-O and C═O groups responded first under the disturbance of sulfamethoxazole. Excessive sulfamethoxazole (20 mg L-1) would decrease the ratio of α-helix/(ß-sheet + random coil) of extracellular proteins, resulting in a loose structure. Molecular docking and dynamic simulation revealed that extracellular proteins would provide abundant sites to bind with sulfamethoxazole, through hydrogen bond and Pi-Akyl hydrophobic interaction forces. Once sulfamethoxazole penetrates into the cell surface and combines with the transmembrane ammonium transport domain, it may inhibit the NH4+ transport. Our findings enhance the understanding on the interaction of extracellular proteins and sulfamethoxazole, which may be valuable for deciphering the response property of anammox sludge under the antibiotic stress.

Extracellular polymeric substances excreted by anammox sludge act as a barrier for As(III) invasion: Binding property and interaction mechanism.

The arsenic in livestock wastewater would induce adverse impact on the biological treatment technology such as anaerobic ammonium oxidation (anammox) process. Extracellular polymeric substances (EPS) play an important role in resisting such toxicity. Unfortunately, the role of EPS in protecting anammox from As(III) and the mechanisms underlying the protection still remains unclear. This work comprehensively evaluated the acute toxicity of arsenic on anammox sludge and investigated the binding property and interaction mechanism. The results revealed that the half maximal inhibitory concentration (IC50) of As(III) on anammox sludge was estimated to be 408 mg L-1, which decreased to 41.97 mg L-1 when EPS was exfoliated. Complexation and hydrophobic interactions were the leading forces in preventing arsenic invasion. Protein was the main component that complexes with As(III), and O-H, -NH, -CO were binding sites. The response sequence of organic component in EPS to As(III) was ordered as hydrocarbons-proteins-polysaccharides-aliphatic amines.

Comparison of the dynamic responses of different anammox granules to copper nanoparticle stress: Antibiotic exposure history made a difference.

Two types of anaerobic ammonium oxidation (anammox) seed sludge were selected to evaluate their responses to copper nanoparticles (CuNPs) exposure. Antibiotic-exposed anammox granules (R1) were more likely to be inhibited by 5.0 mg L-1 CuNPs than the normal anammox granules (C1). The nitrogen removal efficiency (NRE) of C1 decreased by 9.00% after two weeks of exposure to CuNPs, whereas that of R1 decreased by 20.32%. Simultaneously, the abundance of Candidatus. Kuenenia decreased by 27.65% and 36.02% in C1 and R1 under CuNPs stress conditions, respectively. Generally, R1 was more susceptible to CuNPs than C1. The correlation analysis indicated that the horizontal transfer of antibiotic resistance genes and copA triggered by intI1 facilitated the generation of multiresistance in the anammox process. Moreover, the potential multiresistance mechanism of anammox bacteria was hypothesized based on previous results. The results will generate new ideas for the treatment of complex wastewater using the anammox process.

Multiple electron acceptor-mediated sulfur autotrophic denitrification: Nitrogen source competition, long-term performance and microbial community evolution.

Sulfur-driven autotrophic denitrification (SDAD) is feasible for the treatment of low-C/N-ratio and sulfur-laden wastewaters. The nitrite accumulated in SDAD will affect the performance and stability of the system but can be a potential electron acceptor. Thus, single- and multiple-electron acceptor-mediated SDAD systems were investigated. Batch assays revealed that nitrite and nitrate were the preferential options in the SDAD system with single and multiple electron acceptors, respectively. Synchronous nitrogen and sulfur removal was successfully achieved in continuous flow experiments with multiple electron acceptors, and the system could adapt well to high concentrations of sulfide, nitrate and nitrite (i.e., 720, 108 and 64.8 mg L-1, respectively), with the predominant genera shifting from Thiobacillus (48.88%) at the initial stage to unclassified_p_Firmicute (34.24%) and Syner-01 (12.31%) at the last stage. This work provides a fundamental basis for applying and regulating SDAD with multiple electron acceptors for the remediation of nitrogen- and sulfide- laden wastewaters.

Universal Method to Fabricate Transition Metal Single-Atom-Anchored Carbon with Excellent Oxygen Reduction Reaction Activity.

Single-atom catalysts (SACs) have attracted great attention due to their high atom-utilization and catalytic efficiency. However, a universal synthetic route is still lacking, which restricts the SAC-related investigation and application. Here, we report a simple and cost-effective method to fabricate transition metal SACs through ion exchange and annealing procedures. Benefiting from the "egg-box" structure property of alginate, the metal ion can be effectively anchored into the organic center. Using CuCl2 as a representative transition metal ion, the Cu SAC structure was synthesized and identified by aberration-corrected high-angle annular dark-field scanning transmission electron microscopy and X-ray absorption fine structure spectroscopy. Through optimizing CuCl2 concentration, the obtained Cu SAC exhibited a good oxygen reduction reaction activity, whose onset potential, half wave potential, and limiting current density are all comparable to those of 20 wt % Pt/C. Cu-N4 was identified as the responsible catalytic site. More importantly, other transition metal SACs can be easily synthesized via altering metallic solution, which proves the universality of our proposed method. This work may be valuable for the cost-effective and universal SAC synthetic method development.

Anammox Granules Acclimatized to Mainstream Conditions Can Achieve a Volumetric Nitrogen Removal Rate Comparable to Sidestream Systems.

The implementation of mainstream anammox has gained increasing attention. In this study, the feasibility of using sidestream anammox granules to start up mainstream reactors was investigated by comparing two switching strategies. A maximum nitrogen removal potential of 3.6 ± 0.2 kg N m-3 d-1 was obtained for the reactor after direct switching to mainstream conditions (70 mg TN L-1, 15 °C). Nevertheless, the reactor preacclimatized to 25 °C (Ma) exhibited a higher nitrogen removal potential of 7.0 ± 0.3 kg N m-3 d-1 at 15 °C, which is the highest volumetric nitrogen removal rate of mainstream anammox reactors to date. Candidatus Kuenenia stuttgartiensis was identified as the dominant anammox bacterium, and its relative abundance in two reactors remained stable throughout the whole operation (200 days). Moreover, with the aid of acclimatization, the activation energy was reduced and the specific growth rate became higher. These results indicated that the physiological evolution of the dominant anammox bacterium instead of interspecies selection was the main reason for the high potential during the switch to mainstream conditions. Therefore, using sidestream anammox granules as seed sludge to start up mainstream reactors was demonstrated to be feasible, and a switching strategy of acclimatization at 25 °C was recommended.

Exogenous extracellular polymeric substances as protective agents for the preservation of anammox granules.

The preservation of anammox granules is of great significance for the rapid start-up of the anammox process and improvement of performance stability. Therefore, it is necessary to explore an economical and stable preservation strategy. Exogenous extracellular polymeric substances (EPS) were used as protective agents for the preservation of anammox granules in this study. In brief, EPS from anammox sludge (A-EPS) and denitrifying sludge (D-EPS) were added to preserve anammox sludge at 4 °C and room temperature (15-20 °C). The results showed that A-EPS addition at 4 °C was the optimal condition for the preservation of anammox granules. After 90 days of preservation, the specific anammox activity (SAA) of the anammox granules remained at 92.7 ± 2.2 mg N g-1 VSS day-1 (remaining ratio of 33.4%), while that of the sludge with D-EPS addition at the same temperature was only 77.1 ± 3.2 mg N g-1 VSS day-1 (remaining ratio of 27.8%). The nitrogen removal efficiency of the experimental group with D-EPS at room temperature was 85.9%, and that of the A-EPS group reached 90.6% under the same temperature conditions. The abundance of the functional genes hzsA, hdh and nirS of the sludge (4 °C; A-EPS addition) after recovery were 138.5%, 317.1%, and 375.9%, respectively, of those of sludge from the D-EPS-added group at the same temperature. RDA revealed the contribution of proteins to the preservation process. Overall, this study provides an economical and robust strategy for the preservation of anammox granules.

Advances and challenges of mainstream nitrogen removal from municipal wastewater with anammox-based processes.

Anaerobic ammonium oxidation (anammox) is a novel process of deammonification that exhibits superior ecological and economic potential compared to that of traditional heterotrophic processes. Although this process has been successfully implemented in treating high-strength nitrogen-contaminated wastewater, it still faces many challenges in treating mainstream municipal wastewater. This review aims to provide an overview of the status and challenges of mainstream anammox-based processes. The different configurations and crucial factors are discussed in this review. Finally, the future needs for feasible application are stated. PRACTITIONER POINTS: Factors restricting mainstream application of anammox-based processes are reviewed. Control strategies for selecting and maintaining anammox bacteria are discussed. Recent advances in nitrite production via partial nitrification or denitrification are summarized. Future needs for the feasible application of anammox-based nitrogen removal technology for mainstream municipal wastewater treatment are outlined.

A novel strategy for anammox consortia preservation: Transformation into anoxic sulfide oxidation consortia.

The preservation of anaerobic ammonia oxidation (anammox) consortia is crucial for the rapid start-up and the process stability of the anammox based bioreactor. This work proposed and evaluated the feasibility of an anammox consortia preservation strategy, in which the anammox sludge was transformed into intermediate anoxic sulfide oxidation (ASO) functional microorganisms. Initially, the ASO process was successfully started up by inoculating anammox sludge and the overall sulfide and nitrate removal rates stabilized at 57.5 ± 0.22 and 10.0 ± 0.18 kg m-3 day-1, respectively. Then, the bioreactor function was reversely transformed into anammox, whose nitrogen removal rate reached 1.68 kg m-3 day-1. Granule characteristics analysis revealed that both biomass and extracellular polymeric substance content returned to their original states after the reverse start-up. Although the population of Candidatus_Kuenenia was greatly declined during ASO process, its richness was successfully recovered after the reverse start-up of the anammox process. The inferred metagenomes analysis demonstrated that the shifts in functional microorganisms were related to variation in the main metabolic pathways. The specific activities of anammox and ASO both are regarded as key indicators for the successful start-up of bioreactor. This work revealed a novel technique for the preservation of anammox consortia and might be a potential strategy for overcoming the drawback of long start-up time.