Superior mainstream partial nitritation in an acidic membrane-aerated biofilm reactor.

Shortcut nitrogen removal holds significant economic appeal for mainstream wastewater treatment. Nevertheless, it is too difficult to achieve the stable suppression of nitrite-oxidizing bacteria (NOB), and simultaneously maintain the activity of ammonia-oxidizing bacteria (AOB). This study proposes to overcome this challenge by employing the novel acid-tolerant AOB, namely "Candidatus Nitrosoglobus", in a membrane-aerated biofilm reactor (MABR). Superior partial nitritation was demonstrated in low-strength wastewater from two aspects. First, the long-term operation (256 days) under the acidic pH range of 5.0 to 5.2 showed the successful NOB washout by the in situ free nitrous acid (FNA) of approximately 1 mg N/L. This was evidenced by the stable nitrite accumulation ratio (NAR) close to 100 % and the disappearance of NOB shown by 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization. Second, oxygen was sufficiently supplied in the MABR, leading to an unprecedentedly high ammonia oxidation rate (AOR) at 2.4 ± 0.1 kg N/(m3 d) at a short hydraulic retention time (HRT) of a mere 30 min. Due to the counter diffusion of substrates, the present acidic MABR displayed a significantly higher apparent oxygen affinity (0.36 ± 0.03 mg O2/L), a marginally lower apparent ammonia affinity (14.9 ± 1.9 mg N/L), and a heightened sensitivity to FNA and pH variations, compared with counterparts determined by flocculant acid-tolerant AOB. Beyond supporting the potential application of shortcut nitrogen removal in mainstream wastewater, this study also offers the attractive prospect of intensifying wastewater treatment by markedly reducing the HRT of the aerobic unit.

Single cell protein production from methane in a gas-delivery membrane bioreactor.

Single cell protein (SCP, or microbial protein) is one of the emerging alternative protein sources to address the global challenge of food insecurity. Recently, the SCP produced from methane has attracted substantial attention since methane is a renewable resource attainable from anaerobic digestion. However, the supply of methane, an insoluble gas in water, is one of the major challenges in producing methane-based SCP. This work developed a novel bioreactor configuration, in which hollow fiber membrane was used for efficient methane supply while microorganisms were growing in the suspended form favourable for the biomass harvest. Over a 312-day operation, the impacts of three critical parameters on the SCP production were investigated, including the ratio of methane loading to ammonium loading, the ratio of methane loading to oxygen loading, and the sludge retention time (SRT). Under the condition of 4 g CH4/g NH4+, 4 g O2/g CH4, and SRT of 4 days, the highest SCP production yield was observed and determined to be 1.36 g SCP/g CH4 and 5.05 g SCP/g N, respectively. The protein content was up to 67 %, which is higher than the majority of reported values to date. Moreover, the methane and ammonium utilization efficiencies were both close to 100 %, suggesting the highly efficient utilization of substrates in this new bioreactor configuration. A high relative abundance of essential amino acids (EAA) above 42 % was achieved, representing the highest EAA content reported. These findings provide valuable insights into SCP production using methane as a feedstock.

Ammonium-based bioleaching of toxic metals from sewage sludge in a continuous bioreactor.

The broader reuse of sewage sludge as a soil fertilizer or conditioner is impeded by the presence of toxic metals. Bioleaching, a process that leverages microbial metabolisms and metabolites for metal extraction, is viewed as an economically and environmentally feasible approach for metal removal. This study presents an innovative bioleaching process based on microbial oxidation of ammonia released from sludge hydrolysis, mediated by a novel acid tolerant ammonia-oxidizing bacteria (AOB), Ca. Nitrosoglobus. Over a span of 1024 days, a laboratory-scale bioleaching reactor processing anaerobically digested (AD) sludge achieved an in-situ pH of 2.5 ± 0.3. This acidic environment facilitated efficient leaching of toxic metals from AD sludge, upgrading its quality from Grade C to Grade A (qualified for unrestricted use), according to both stabilization and contaminants criteria. The improved quality of AD sludge could potentially reduce sludge disposal expenses and enable a broader reuse of biosolids. Furthermore, this study revealed a pH-dependent total ammonia affinity of Ca. Nitrosoglobus, with a higher affinity constant at pH 3.5 (67.3 ± 20.7 mg N/L) compared to pH 4.5-7.5 (7.6 - 9.6 mg N/L). This finding indicates that by optimizing ammonium concentrations, the efficiency of this novel ammonium-based bioleaching process could be significantly increased.

Coupling Partial Nitritation, Anammox and n-DAMO in a membrane aerated biofilm reactor for simultaneous dissolved methane and nitrogen removal.

Anaerobic technologies with downstream autotrophic nitrogen removal have been proposed to enhance bioenergy recovery and transform a wastewater treatment plant from an energy consumer to an energy exporter. However, approximately 20-50 % of the produced methane is dissolved in the anaerobically treated effluent and is easily stripped into the atmosphere in the downstream aerobic process, contributing to the release of greenhouse gas emissions. This study aims to develop a solution to beneficially utilize dissolved methane to support high-level nitrogen removal from anaerobically treated mainstream wastewater. A novel technology, integrating Partial Nitritation, Anammox and Methane-dependent nitrite/nitrate reduction (i.e. PNAM) was demonstrated in a membrane-aerated biofilm reactor (MABR). With the feeding of ∼50 mg NH4+-N/L and ∼20 mg/L dissolved methane at a hydraulic retention time of 15 h, around 90 % of nitrogen and ∼100 % of dissolved methane can be removed together in the MABR. Microbial community characterization revealed that ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), anammox bacteria, nitrite/nitrate-dependent anaerobic methane oxidation microorganisms (n-DAMO bacteria and archaea) and aerobic methanotrophs co-existed in the established biofilm. Batch tests confirmed the active microbial pathways and showed that AOB, anammox bacteria and n-DAMO microbes were jointly responsible for the nitrogen removal, and dissolved methane was mainly removed by the n-DAMO process, with aerobic methane oxidation making a minor contribution. In addition, the established system was robust against dynamic changes in influent composition. The study provides a promising technology for the simultaneous removal of dissolved methane and nitrogen from domestic wastewater, which can support the transformation of wastewater treatment from an energy- and carbon-intensive process, to one that is energy- and carbon-neutral.

Nitrate-driven anaerobic oxidation of ethane and butane by bacteria.

The short-chain gaseous alkanes (ethane, propane, and butane; SCGAs) are important components of natural gas, yet their fate in environmental systems is poorly understood. Microbially mediated anaerobic oxidation of SCGAs coupled to nitrate reduction has been demonstrated for propane, but is yet to be shown for ethane or butane-despite being energetically feasible. Here we report two independent bacterial enrichments performing anaerobic ethane and butane oxidation, respectively, coupled to nitrate reduction to dinitrogen gas and ammonium. Isotopic 13C- and 15N-labelling experiments, mass and electron balance tests, and metabolite and meta-omics analyses collectively reveal that the recently described propane-oxidizing "Candidatus Alkanivorans nitratireducens" was also responsible for nitrate-dependent anaerobic oxidation of the SCGAs in both these enrichments. The complete genome of this species encodes alkylsuccinate synthase genes for the activation of ethane/butane via fumarate addition. Further substrate range tests confirm that "Ca. A. nitratireducens" is metabolically versatile, being able to degrade ethane, propane, and butane under anoxic conditions. Moreover, our study proves nitrate as an additional electron sink for ethane and butane in anaerobic environments, and for the first time demonstrates the use of the fumarate addition pathway in anaerobic ethane oxidation. These findings contribute to our understanding of microbial metabolism of SCGAs in anaerobic environments.

The effect of green infrastructure on resilience performance in combined sewer systems under climate change.

Climate change is currently reshaping precipitation patterns, intensifying extremes, and altering runoff dynamics. Particularly susceptible to these impacts are combined sewer systems (CSS), which convey both stormwater and wastewater and can lead to combined sewer overflow (CSO) discharges during heavy rainfall. Green infrastructure (GI) can help mitigate these discharges and enhance system resilience under historical conditions; however, the quantification of its effect on resilience in a future climate remains unknown in the literature. This study employs a modified Global Resilience Analysis (GRA) framework for continuous simulation to quantify the impact of climate change on CSS resilience, particularly CSOs. The study assesses the efficacy of GI interventions (green roofs, permeable pavements, and bioretention cells) under diverse future rainfall scenarios based on EURO-CORDEX regional climate models (2085-2099) and three Representative Concentration Pathways (2.6, 4.5, 8.5 W/m2). The findings underscore a general decline in resilience indices across the future rainfall scenarios considered. Notably, the total yearly CSO discharge volume increases by a range of 145 % to 256 % in response to different rainfall scenarios. While GI proves effective in increasing resilience, it falls short of offsetting the impacts of climate change. Among the GI options assessed, green roofs routed to pervious areas exhibit the highest adaptive capacity, ranging from 9 % to 22 % at a system level, followed by permeable pavements with an adaptation capacity between 7 and 13 %. By linking the effects of future rainfall scenarios on CSO performance, this study contributes to understanding GI's potential as a strategic tool for enhancing urban resilience.

Effect of tetrahedral framework nucleic acids on the reconstruction of tendon-to-bone injuries after rotator cuff tears.

Clinicians and researchers have always faced challenges in performing surgery for rotator cuff tears (RCT) due to the intricate nature of the tendon-bone gradient and the limited long-term effectiveness. At the same time, the occurrence of an inflammatory microenvironment further aggravates tissue damage, which has a negative impact on the regeneration process of mesenchymal stem cells (MSCs) and eventually leads to the production of scar tissue. Tetrahedral framework nucleic acids (tFNAs), novel nanomaterials, have shown great potential in biomedicine due to their strong biocompatibility, excellent cellular internalisation ability, and unparalleled programmability. The objective of this research was to examine if tFNAs have a positive effect on regeneration after RCTs. Experiments conducted in a controlled environment demonstrated that tFNAs hindered the assembly of inflammasomes in macrophages, resulting in a decrease in the release of inflammatory factors. Next, tFNAs were shown to exert a protective effect on the osteogenic and chondrogenic differentiation of bone marrow MSCs under inflammatory conditions. The in vitro results also demonstrated the regulatory effect of tFNAs on tendon-related protein expression levels in tenocytes after inflammatory stimulation. Finally, intra-articular injection of tFNAs into a rat RCT model showed that tFNAs improved tendon-to-bone healing, suggesting that tFNAs may be promising tendon-to-bone protective agents for the treatment of RCTs.

Humic substances as electron acceptor for anaerobic oxidation of methane (AOM) and electron shuttle in Mn (IV)-dependent AOM.

Anaerobic methanotrophic archaea (ANME) belonging to the family Methanoperedenaceae are crucial for the global carbon cycle and different biogeochemical processes, owing to their metabolic versatility to couple anaerobic oxidation of methane (AOM) with different electron acceptors. A universal feature of Methanoperedenaceae is the abundant genes encoded in their genomes associated with extracellular electron transfer (EET) pathways. Candidatus. 'Methanoperedens manganicus', an archaeon belonging to the family Methanoperedenaceae, was recently enriched in a bioreactor performing AOM coupled with Mn (IV) reduction. Using this EET-capable ANME, we tested the hypothesis in this study that ANME can catalyse the humic-dependent AOM for growth. A two-year incubation showed that AOM activity can be sustained by Ca. 'M. manganicus' consortium in a bioreactor fed only with humic acids and methane. An isotopic mass balance batch test confirmed that the observed AOM was coupled to the reduction of humic acids. The increase of relative abundance of Ca. 'M. manganicus', and the total archaea population in the microbial community suggested that Ca. 'M. manganicus' can grow on methane and humic acids. The observation of humic-dependent AOM led to a subsequent hypothesis that humic acids could be used as the electron shuttle to mediate the EET in dissimilatory Mn (IV) reduction by Ca. 'M. manganicus'. We tested this hypothesis by adding humic acids to a Ca. 'M. manganicus' dominated-culture, which showed that the AOM rate was doubled by the addition of humic acids. X-ray photoelectron spectroscopy (XPS) showed that quinone moieties were consumed when humic acids worked as electron acceptors while remaining stable when functioning as a shuttle for electron transfer. The results of our study suggest that humic acids may serve as electron shuttles to allow ANME to access more electron acceptors through long-range EET.

Pyrogenic Carbon Promotes Anaerobic Oxidation of Methane Coupled with Iron Reduction via the Redox-Cycling Mechanism.

Pyrogenic carbon (PC) can mediate electron transfer and thus catalyze biogeochemical processes to impact greenhouse gas (GHG) emissions. Here, we demonstrate that PC can contribute to mitigating GHG emissions by promoting the Fe(III)-dependent anaerobic oxidation of methane (AOM). It was found that the amendment PCs in microcosms dominated by Methanoperedenaceae performing Fe(III)-dependent AOM simultaneously promoted the rate of AOM and Fe(III) reduction with a consistent ratio close to the theoretical stoichiometry of 1:8. Further correlation analysis showed that the AOM rate was linearly correlated with the electron exchange capacity, but not the conductivity, of added PC materials, indicating the redox-cycling electron transfer mechanism to promote the Fe(III)-dependent AOM. The mass content of the C═O moiety from differentially treated PCs was well correlated with the AOM rate, suggesting that surface redox-active quinone groups on PCs contribute to facilitating Fe(III)-dependent AOM. Further microbial analyses indicate that PC likely shuttles direct electron transfer from Methanoperedenaceae to Fe(III) reduction. This study provides new insight into the climate-cooling impact of PCs, and our evaluation indicates that the PC-facilitated Fe(III)-dependent AOM could have a significant contribution to suppressing methane emissions from the world's reservoirs.

Multifaceted benefits of magnesium hydroxide dosing in sewer systems: Impacts on downstream wastewater treatment processes.

Magnesium hydroxide [Mg(OH)2] is a non-hazardous chemical widely applied in sewer systems for managing odour and corrosion. Despite its proven effectiveness in mitigating these issues, the impacts of dosing Mg(OH)2 in sewers on downstream wastewater treatment plants have not been comprehensively investigated. Through a one-year operation of laboratory-scale urban wastewater systems, including sewer reactors, sequencing batch reactors, and anaerobic sludge digesters, the findings indicated that Mg(OH)2 dosing in sewer systems had multifaceted benefits on downstream treatment processes. Compared to the control, the Mg(OH)2-dosed experimental system displayed elevated sewage pH (8.8±0.1vs 7.1±0.1), reduced sulfide concentration by 35.1%±4.9% (6.7±0.9mgSL-1), and lower methane concentration by 58.0%±4.9% (19.1±3.6mgCODL-1). Additionally, it increased alkalinity by 16.3%±2.2% (51.9±5.4mgCaCO3L-1), and volatile fatty acids concentration by 207.4%±22.2% (56.6±9.0mgCODL-1) in sewer effluent. While these changes offered limited advantages for downstream nitrogen removal in systems with sufficient alkalinity and carbon sources, significant improvements in ammonium oxidation rate and NOx reduction rate were observed in cases with limited alkalinity and carbon sources availability. Moreover, Mg(OH)2 dosing in upstream did not have any detrimental effects on anaerobic sludge digesters. Magnesium-phosphate precipitation led to a 31.7%±4.1% reduction in phosphate concertation in anaerobic digester sludge supernatant (56.1±10.4mgPL-1). The retention of magnesium in sludge increased settleability by 13.9%±1.6% and improved digested sludge dewaterability by 10.7%±5.3%. Consequently, the use of Mg(OH)2 dosing in sewers could potentially reduce downstream chemical demand and costs for carbon sources (e.g., acetate), pH adjustment and sludge dewatering.

Challenges of suppressing nitrite-oxidizing bacteria in membrane aerated biofilm reactors by low dissolved oxygen control.

Membrane aerated biofilm reactor (MABR) and shortcut nitrogen removal are two types of solutions to reduce energy consumption in wastewater treatment, with the former improving the aeration efficiency and the latter reducing the oxygen demand. However, integrating these two solutions, i.e., achieving shortcut nitrogen removal in MABR, is challenging due to the difficulty in suppressing nitrite-oxidizing bacteria (NOB). In this study, four MABRs were established to demonstrate the feasibility of initiating, maintaining, and restoring NOB suppression using low dissolved oxygen (DO) control, in the presence and absence of anammox bacteria, respectively. Long-term results revealed that the strict low DO (< 0.1 mg/L) in MABR could initiate and maintain stable NOB suppression for more than five months with nitrite accumulation ratio above 90 %, but it was unable to re-suppress NOB once they prevailed. Moreover, the presence of anammox bacteria increased the threshold of DO level to maintain NOB suppression in MABRs, but it was still incapable to restore the deteriorated NOB suppression in conjunction with low DO control. Mathematical modelling confirmed the experimental results and further explored the differences of NOB suppression in conventional biofilms and MABR biofilms. Simulation results showed that it is more challenging to maintain stable NOB suppression in MABRs compared to conventional biofilms, regardless of biofilm thickness or influent nitrogen concentration. Kinetic mechanisms for NOB suppression in different types of biofilms were proposed, suggesting that it is difficult to wash out NOB developed in the innermost layer of MABR biofilms because of the high oxygen level and low sludge wasting rate. In summary, this study systematically demonstrated the challenges of NOB suppression in MABRs through both experiments and mathematical modelling. These findings provide valuable insights into the applications of MABRs and call for more studies in developing effective strategies to achieve stable shortcut nitrogen removal in this energy-efficient configuration.

Integrated urban water management by coupling iron salt production and application with biogas upgrading.

Integrated urban water management is a well-accepted concept for managing urban water. It requires efficient and integrated technological solutions that enable system-wide gains via a whole-of-system approach. Here, we create a solid link between the manufacturing of an iron salt, its application in an urban water system, and high-quality bioenergy recovery from wastewater. An iron-oxidising electrochemical cell is used to remove CO2 (also H2S and NH3) from biogas, thus achieving biogas upgrading, and simultaneously producing FeCO3. The subsequent dose of the electrochemically produced FeCO3 to wastewater and sludge removes sulfide and phosphate, and enhances sludge settleability and dewaterability, with comparable or superior performance compared to the imported and hazardous iron salts it substitutes (FeCl2, and FeCl3). The process enables water utilities to establish a self-reliant and more secure supply chain to meet its demand for iron salts, at lower economic and environmental costs, and simultaneously achieve recovery of high-quality bioenergy.

Multi-heme cytochrome-mediated extracellular electron transfer by the anaerobic methanotroph 'Candidatus Methanoperedens nitroreducens'.

Anaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by 'Candidatus Methanoperedens nitroreducens', an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that 'Ca. M. nitroreducens' uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used.

Decoupling the simultaneous effects of NO2-, pH and free nitrous acid on N2O and NO production from enriched nitrifying activated sludge.

In the pursuit of energy and carbon neutrality, nitrogen removal technologies have been developed featuring nitrite (NO2-) accumulation. However, high NO2- accumulations are often associated with stimulated greenhouse gas (i.e., nitrous oxide, N2O) emissions. Furthermore, the coexistence of free nitrous acid (FNA) formed by NO2- and proton (pH) makes the consequence of NO2- accumulation on N2O emissions complicated. The concurrent three factors, NO2-, pH and FNA may play different roles on N2O and nitric oxide (NO) emissions simultaneously, which has not been systematically studied. This study aims to decouple the effects of NO2- (0-200 mg N/L), pH (6.5-8) and FNA (0-0.15 mg N/L) on the N2O and NO production rates and the production pathways by ammonia oxidizing bacteria (AOB), with the use of a series of precisely executed batch tests and isotope site-preference analysis. Results suggested the dominant factors affecting the N2O production rate were NO2- and FNA concentrations, while pH alone played a relatively insignificant role. The most influential factor shifted from NO2- to FNA as FNA concentrations increased from 0 to 0.15 mg N/L. At concentrations below 0.0045 mg HNO2-N/L, nitrite rather than FNA played a significant role stimulating N2O production at elevated nitrite concentrations. The inhibition effect of FNA emerged with further increase of FNA between 0.0045-0.015 mg HNO2-N/L, weakening the promoting effect of increased nitrite. While at concentrations above 0.015 mg HNO2-N/L, FNA inhibited N2O production especially from nitrifier denitrification pathway with the level of inhibition linearly correlated with the FNA concentration. pH and the nitrite concentration regulated the production pathways, with elevated pH promoting the nitrifier nitrification pathway, while elevated NO2- concentrations promoting the nitrifier denitrification pathway. In contrast to N2O, NO emission was less susceptible to FNA at concentrations up to 0.015 mg N/L but was stimulated by increasing NO2- concentrations. This study, for the first time, distinguished the effects of pH, NO2- and FNA on N2O and NO production, thereby providing support to the design and operation of novel nitrogen removal systems with NO2- accumulation.

Establishing boundary conditions in sewer pipe/soil heat transfer modelling using physics-informed learning.

Modelling heat transfer in sewers and the surrounding soil is important for effective sewer maintenance, and for heat recovery from wastewater. The boundary conditions, including both the thickness of the soil layer to be modelled and the temperature distribution around the boundary of the soil layer, directly determine both the efficiency and accuracy of the models. Yet there is no systematic method to establish these conditions. This study presents a novel and generic approach to establishing efficient boundary conditions for sewer heat transfer modelling. Fourier transform is applied to identify the dominant frequencies of the temperatures of the heat sources/sinks, namely the atmosphere, sewer air and wastewater. A simple data-driven model for determining the thickness of the soil-layer to be included, and three physics-informed models for predicting the temperatures at the soil-layer boundary are then learnt from mechanistic models for sewer heat transfer, taking into consideration the frequency spectra. The methodology achieved high fidelity to the mechanistic models in predicting the soil-layer boundary temperatures and sewer wall temperatures for real-life sewers. This approach offers an easy yet reliable way to obtain efficient boundary conditions that significantly improve both the accuracy and speed of sewer heat transfer modelling.

Hydrophilic nanofibers with aligned topography modulate macrophage-mediated host responses via the NLRP3 inflammasome.

Successful biomaterial implantation requires appropriate immune responses. Macrophages are key mediators involved in this process. Currently, exploitation of the intrinsic properties of biomaterials to modulate macrophages and immune responses is appealing. In this study, we prepared hydrophilic nanofibers with an aligned topography by incorporating polyethylene glycol and polycaprolactone using axial electrospinning. We investigated the effect of the nanofibers on macrophage behavior and the underlying mechanisms. With the increase of hydrophilicity of aligned nanofibers, the inflammatory gene expression of macrophages adhering to them was downregulated, and M2 polarization was induced. We further presented clear evidence that the inflammasome NOD-like receptor thermal protein domain associated protein 3 (NLRP3) was the cellular sensor by which macrophages sense the biomaterials, and it acted as a regulator of the macrophage-mediated response to foreign bodies and implant integration. In vivo, we showed that the fibers shaped the implant-related immune microenvironment and ameliorated peritendinous adhesions. In conclusion, our study demonstrated that hydrophilic aligned nanofibers exhibited better biocompatibility and immunological properties.

Global resilience analysis of combined sewer systems under continuous hydrologic simulation.

Managing and reducing combined sewer overflow (CSO) discharges is crucial for enhancing the resilience of combined sewer systems (CSS). However, the absence of a standardised resilience analysis approach poses challenges in developing effective discharge reduction strategies. To address this, our study presents a top-down method that expands the existing Global Resilience Analysis to quantify resilience performance in CSS. This approach establishes a link between threats (e.g., rainfall) and impacts (e.g., CSOs) through continuous and long-term simulation, accommodating various rainfall patterns, including extreme events. We assess CSO discharge impacts from a resilience perspective by introducing eight new metrics. We conducted a case study in Fehraltorf, Switzerland, analysing the performance of three green infrastructure (GI) types (bioretention cells, green roofs, and permeable pavements) over 38 years. The results demonstrated that GI enhanced all resilience indices, with variations observed in individual CSO performance metrics and their system locations. Notably, in Fehraltorf, green roofs emerged as the most effective GI type for improving resilience, while the downstream outfall displayed the highest resilience enhancement. Overall, our proposed method enables a shift from event-based to continuous simulation analysis, providing a standardised approach for resilience assessment. This approach informs the development of strategies for CSO discharge reduction and the enhancement of CSS resilience.

Adaptation of anammox process for nitrogen removal from acidic nitritation effluent in a low pH moving bed biofilm reactor.

Acidic partial nitritation (PN) has emerged to be a promisingly stable process in wastewater treatment, which can simultaneously achieve nitrite accumulation and about half of ammonium reduction. However, directly applying anaerobic ammonium oxidation (anammox) process to treat the acidic PN effluent (pH 4-5) is susceptible to the inhibition of anammox bacteria. Here, this study demonstrated the adaptation of anammox process to acidic pH in a moving bed biofilm reactor (MBBR). By feeding the laboratory-scale MBBR with acidic PN effluent (pH = 4.6 ± 0.2), the pH of an anammox reactor was self-sustained in the range of pH 5 - 6. Yet, a high total nitrogen removal efficiency of over 80% at a practical loading rate of up to 149.7 ± 3.9 mg N/L/d was achieved. Comprehensive microbial assessment, including amplicon sequencing, metagenomics, cryosection-FISH, and qPCR, identified that Candidatus Brocadia, close to known neutrophilic members, was the dominant anammox bacteria. Anammox bacteria were found present in the inner layer of thick biofilms but barely present in the surface layer of thick biofilms and in thin biofilms. Results from batch tests also showed that the activity of anammox biofilms could be maintained when subjected to pH 5 at a nitrite concentration of 10 mg N/L, whereas the activity was completely inhibited after disturbing the biofilm structure. These results collectively indicate that the anammox bacteria enriched in the present acidic MBBR could not be inherently acid-tolerant. Instead, the achieved stable anammox performance under the acidic condition is likely due to biofilm stratification and protection. This result highlights the biofilm configuration as a useful solution to address nitrogen removal from acidic PN effluent, and also suggests that biofilm may play a critical role in protecting anammox bacteria found in many acidic nature environments.

Insights on biological phosphorus removal with partial nitrification in single sludge system via sidestream free ammonia and free nitrous acid dosing.

The sidestream sludge treatment by free ammonium (FA)/free nitrous acid (FNA) dosing was frequently demonstrated to maintain the nitrite pathway for the partial nitrification (PN) process. Nevertheless, the inhibitory effect of FA and FNA would severely influence polyphosphate accumulating organisms (PAOs), destroying the microbe-based phosphorus (P) removal. Therefore, a strategic evaluation was proposed to successfully achieve biological P removal with a partial nitrification process in a single sludge system by sidestream FA and FNA dosing. Through the long-term operation of 500 days, excellent phosphorus, ammonium and total nitrogen removal performance were achieved at 97.5 ± 2.6 %, 99.1 ± 1.0 % and 75.5 ± 0.4 %, respectively. Stable partial nitrification with a nitrite accumulation ratio (NAR) of 94.1 ± 3.4 was attained. The batch tests also reported the robust aerobic phosphorus uptake based on FA and FNA adapted sludge after exposure of FA and FNA, respectively, suggesting the FA and FNA treatment strategy could potentially offer the opportunity for the selection of PAOs, which synchronously have the tolerance to FA and FNA. Microbial community analysis suggested that Accumulibacter, Tetrasphaera, and Comamonadaceae collectively contributed to the phosphorus removal in this system. Summarily, the proposed work presents a novel and feasible strategy to integrate enhanced biological phosphorus removal (EBPR) and short-cut nitrogen cycling and bring the combined mainstream phosphorus removal and partial nitrification process closer to practical application.

Salinity effect on an anaerobic methane- and ammonium-oxidising consortium: Shifts in activity, morphology, osmoregulation and syntrophic relationship.

Nitrate-dependent anaerobic methane oxidation (AOM) is a microbial process of both ecological significance for global methane mitigation and application potential for wastewater treatment. It is mediated by organisms belonging to the archaeal family 'Candidatus Methanoperedenaceae', which have so far mainly been found in freshwater environments. Their potential distribution in saline environments and their physiological responses to salinity variation were still poorly understood. In this study, the responses of the freshwater 'Candidatus Methanoperedens nitroreducens'-dominated consortium to different salinities were investigated using short- and long-term setups. Short-term exposure to salt stress significantly affected nitrate reduction and methane oxidation activities over the tested concentration range of 15-200‰ NaCl, and 'Ca. M. nitroreducens' showed the higher tolerance to high salinity stress than its partner of anammox bacteria. At high salinity concentration, near marine conditions of 37‰, the target organism 'Ca. M. nitroreducens' showed stabilized nitrate reduction activity of 208.5 µmol day-1 gCDW-1 in long-term bioreactors over 300 days, in comparison to 362.9 and 334.3 µmol day-1 gCDW-1 under low-salinity conditions (1.7‰ NaCl) and control conditions (∼15‰ NaCl). Different partners of 'Ca.  M. nitroreducens' evolved in the consortia with three different salinity conditions, suggesting the different syntrophic mechanisms shaped by changes in salinity. A new syntrophic relationship between 'Ca. M. nitroreducens' and Fimicutes and/or Chloroflexi denitrifying populations was identified under the marine salinity condition. Metaproteomic analysis shows that the salinity changes lead to higher expression of response regulators and selective ion (Na+/H+) channeling proteins that can regulate the osmotic pressure between the cell and its environment. The reverse methanogenesis pathway was, however, not impacted. The finding of this study has important implications for the ecological distribution of the nitrate-dependent AOM process in marine environments and the potential of this biotechnological process for the treatment of high-salinity industrial wastewater.