Abstract Mainstream anammox implementation for nitrogen removal is constrained by unstable nitrite supply and organic carbon requirements for nitrate byproduct removal. This study evaluated integrated fixed-film activated sludge (IFAS) biofilms to enhance anammox activity by coupling low-oxygen ammonium oxidation with volatile fatty acid (VFA)-driven nitrate reduction. Nanopore long-read metagenomic assembly recovered a high-quality, circular single-contig Candidatus Brocadia sapporoensis metagenome-assembled genome (MAG) from full-scale IFAS biofilms. This MAG encodes complete anammox metabolism, dissimilatory nitrate reduction to ammonium (DNRA) and acetate/propionate carbon transformation pathways. Metatranscriptomics showed that low dissolved oxygen (DO) upregulated Ca. B. sapporoensis genes involved in anammox, nitrate reduction, and carbon metabolism. Microaerobic assays established a DO level of 0.7 mg/L as optimal for sustaining near-maximal ammonium oxidation alongside anammox-driven total inorganic nitrogen (TIN) loss. Anoxic tests conducted in secondary effluent indicated that external acetate amendment promoted greater partial nitrate reduction and TIN loss than additional propionate amendment. Integrating this dissolved oxygen concentration with external acetate amendment in a two-stage microaerobic–anoxic system successfully achieved sequential ammonium oxidation, partial nitrate reduction, and anammox-mediated TIN removal. Stage-specific expression suggested Ca. B. sapporoensis could contribute to nitrite self-supplementation via nxrAB -mediated nitrate reduction. Overall, microaerobic ammonium oxidation and Ca. B. sapporoensis-driven partial nitrate reduction jointly sustain mainstream anammox activity. Furthermore, this study demonstrates that successful metabolic synergy depends fundamentally upon precise dissolved oxygen control and effective external acetate amendment.

Abstract Genomovar-level and intragenomic diversity cannot be resolved by conventional amplicon sequencing due to the limitation of fragment lengths and read accuracy, while the application of metagenomic profiling to a large number of samples can be resource intensive. Here, we report UltraRes-rrn, an integrated wet-lab and computational workflow for high-accuracy rrn (i.e., 16S– ITS–23S rRNA) operon profiling using Nanopore sequencing. By integrating ultra-long DNA recovery, long-read amplicon sequencing, and unique molecular identifiers (UMI)-based consensus correction, UltraRes-rrn obtains full-length 16S–ITS–23S rRNA operon consensus sequences with mean accuracies exceeding 99.98%. To achieve higher resolution, we propose a hierarchical rRNA operon profiling strategy in which concatenated 16S+23S rRNA genes (16S23S) serve as a primary and the internal transcribed spacer (ITS) provides a secondary marker. The 16S23S marker achieved discrimination at the genomovar level compared to either 16S or 23S rRNA genes alone, which mitigates the ITS-driven over-splitting observed with the full-length rrn operon and allows for larger proportion of data being classified at higher confidence thresholds. Further, ITS variation was strongly structured by tRNA occurrence patterns, suggesting that ITS can capture taxon microdiversity missed by either 16S or 23S rRNA gene sequences alone. The UltraRes-rrn workflow was applied to full-scale nitrogen removal reactors, revealing intraspecies diversity variation driven by different carbon regimes, which would not have been possible with a shorter gene sequence. In summary, UltraRes-rrn enables cost-effective community profiling at genomovar-level resolution in complex ecosystems.

A field-deployable DNA sequencing approach for quantitative microbial community profiling can enable rapid responses for a range of applications in the water sector-from process control to wastewater surveillance. Current quantitative approaches require complex instrumentation and have long turnaround times for DNA recovery and absolute quantitation. In this study, we report a field-deployable rapid detection and rapid absolute quantitation (rD+rQ) workflow that leverages real-time Nanopore sequencing for quantitative metagenomics. This workflow integrates a high-molecular-weight DNA recovery protocol for diverse environmental matrices of relevance to the water sector, and multiplexed Nanopore sequencing with barcoded spike-in-based calibration (BSINC). BSINC using multispecies genomic spike-in controls exhibits significantly higher calibration accuracy compared to conventional approaches that utilize either a single DNA fragment or single organism spike-in controls. Dynamic detection and quantitation limits were established based on the coverage fraction of sequenced genomes and the coefficient of variation of genome copy numbers across replicates to enhance the accuracy and precision of microbial quantitation. The rD+rQ workflow achieves species-level identification and absolute quantitative results comparable to digital PCR in environmental samples. This portable laboratory and easy-to-use rD+rQ workflow should facilitate rapid decision-making for the water industry.IMPORTANCERapid and real-time monitoring of microbial communities is critical for a vast array of applications in environmental microbiology and biotechnology. While recent developments in portable sequencing technologies and associated workflows make onsite analysis possible, these approaches do not provide quantitative data on microbial concentrations. In this study, we present a sample and data processing workflow that enables nontargeted and quantitative microbial community profiling and demonstrate its validity on complex environmental samples. This approach for acquiring quantitative data can drive rapid decision-making from bioprocess control to wastewater-based epidemiology.

Performic acid (PFA) is receiving increasing attention as a fast-acting disinfectant for municipal wastewater effluents and combined sewer overflows (CSOs), where short contact times and highly variable matrices constrain conventional treatment. However, its practical adoption is limited by intrinsic chemical instability, safety considerations, and dependence on point-of-use generation. This critical review integrates peer-reviewed literature and patent evidence to evaluate how PFA production and delivery architectures govern field deployability. We compare five technology families including on-site precursor blending, distillation and reactive separation, co-delivery formulations, microfluidic on-demand generation, and electrochemical synthesis using structured evidence base and readiness-aware scoring framework. Performance is interpreted through the lens of time-integrated residual exposure, reflecting rapid oxidant decay and matrix-dependent demand. Reported full-scale and pilot studies indicate that PFA can achieve rapid bacterial inactivation at low exposure values, and, in some cases, strong target-dependent viral inactivation, often with lower halogenated by-product formation than chlorination, but outcomes are highly sensitive to mixing, particulate shielding, and early-time oxidant consumption. Comparative analysis shows that on-site blending remains the only widely validated municipal-scale approach, while microreactors and reactive separation offer higher attainable strengths at the expense of scale-up and operational complexity, and electrochemical routes remain constrained by low titers and limited dynamic response. By linking disinfection performance to production logistics, safety, and control requirements, and environmental acceptability, this review provides an exposure-anchored framework for technology selection and identifies priority research needs for advancing PFA toward robust, scalable, and regulation-ready deployment in wastewater and CSO disinfection.

Aeration is a major cost at biological nutrient removal (BNR) plants. We report on microbial communities in a pilot-scale BNR system before and after a dissolved oxygen transition from 2.5 to 0.2 mg/L implemented over 18 months. Four PacBio metagenomes and 316 metagenome-assembled genomes are announced.

While ammonia-based aeration control (ABAC) significantly improves process efficiency in water resource recovery facilities (WRRFs), its performance is often limited by the difficulty of tuning proportional-integral (PI) controllers amidst dynamic loads and nonlinear reaction kinetics. This study proposed a systematic tuning approach that derives first-order plus deadtime (FOPDT) parameters from a reduced-order model based on empirical reactor hydraulics. Furthermore, the nonlinearity of Monod saturation kinetics, which describe the impact of dissolved oxygen (SO2) on nitrification rates, is explicitly integrated into the feedback control structure to linearize the system response. The approach was validated via both a model-based simulator and full-scale implementation. In the model-based simulation, both controller structures provided stable performance, but the direct SO2 controller showed nonlinear overshoot during high load periods, while the inclusion of Monod kinetics in ABAC linearized the response, particularly when tuned with the reduced-order model (mean absolute error (MAE) 0.09 mg N/L). At the full-scale plant, when tuned using the proposed method, the controller demonstrated stable performance and successfully attenuated dynamic loads to achieve a low 0.16 mg N/L MAE. These results demonstrate that combining reduced-order modeling with kinetic-based control structures offers a robust automatable alternative to heuristic tuning methods.

Side-Stream Enhanced Biological Phosphorus Removal (S2EBPR) has emerged as a promising technology addressing certain challenges of conventional Enhanced Biological Phosphorus Removal (EBPR), notably stability in phosphorus removal, yet the underlying mechanisms are not fully understood. Metagenomic analysis presents a powerful approach to elucidate community-level metabolic differences between EBPR and S2EBPR configurations. In this study, we compared three EBPR and three S2EBPR activated sludge communities using metagenomic analysis at taxonomy, key functional pathways/genes, and polyphosphate-metabolism marker genes. Our analysis revealed larger genus-level diversity variance in S2EBPR communities, indicating distinct microbial community compositions influenced by different operational configurations. A higher diversity index in the S2EBPR than the EBPR was observed, and a higher Ca. Accumulibacter abundance was detected in EBPRs, whereas the fermentative candidate PAOs genera, including Ca. Phosphoribacter and Ca. Promineifilum, were more abundant in S2EBPR systems. EBPR and S2EBPR groups displayed similar gene and pathway abundance patterns related to core metabolisms essential for carbon and nitrogen metabolism. PolyP-metabolism marker gene phylogeny analysis suggested that exopolyphosphatase gene (ppx) showed better distinctions between EBPR and S2EBPR communities than polyphosphate kinase gene (ppk). This also highlighted the needs in fine-cale microdiversity analysis and finding novel Ca. Accumulibacter clades and species as resolved using the ppk gene. These findings provide valuable insights into AS community dynamics and metabolic functionalities, paving the way for further research into optimizing phosphorus removal processes in wastewater treatment systems.

In the present study, a novel pilot ozone contactor configuration was employed using hydrogen peroxide (H2O2) and multiple ozone diffusion zones in an over-under contactor for testing three wastewater effluents. With a 1 : 1 molar H2O2 : O3 dose, splitting the ozone dose between three diffusers reduced bromate formation by as much as 93% compared to the traditional single diffuser control condition. The required H2O2 dose for similar bromate levels was decreased by more than 90%. 1,4-Dioxane was used as a representative contaminant and hydroxyl radical (OH) probe compound. H2O2 addition significantly improved 1,4-dioxane removal, and removal was similar between different diffuser conditions for the same total ozone dose. Detailed ozone residual and ozone exposure measurements showed that, with H2O2, similar ozone exposure was provided between the single and multi-diffuser H2O2 experiments. This indicates that minimization of local ozone concentration, rather than exposure, is vital for preventing the O3-Br reaction which controls bromate formation and may be beneficial for removal of ozone reactive contaminants and disinfection. Ozone decay, both with and without H2O2, was extremely sensitive to pH. Bromate formation increased by a factor of nearly two from pH 6 to 8 in the control condition, while the effect was less pronounced with H2O2. 1,4-Dioxane removal was unaffected by pH or temperature, while bromate formation decreased with increasing temperature.

Research on low dissolved oxygen (DO) enhanced biological phosphorus removal (EBPR) at full-scale remains limited, a knowledge gap this study aims to fill by investigating EBPR performance and microbial community shifts at a Water Resource Recovery Facility (WRRF) transitioning to low DO conditions. Average DO concentrations decreased from 2.62 mg O2/L in 2019 to 0.80 mg O2/L in 2023. Simultaneously, average effluent orthophosphate concentrations decreased from 0.57 mg P/L to 0.29 mg P/L, despite the elimination of metal salt addition for chemical precipitation in 2023. Average effluent total phosphorus concentrations remained between 0.47 and 0.67 mg P/L across varying DO concentrations, which reached below 0.50 mg O2/L. Batch tests conducted over a four-year period indicated higher phosphorus release and aerobic uptake rates when full-scale DO concentrations were below 1 mg O2/L. Phosphorus release rates increased from 8.9 ± 1.0 to 12.1 ± 0.6 mg P/g MLVSS/hr, while aerobic phosphorus uptake rates increased from 3.6 ± 0.6 to 5.3 ± 0.4 mg P/g MLVSS/hr. Microbial analysis revealed a community shift toward taxa containing polyphosphate-accumulating organisms (PAOs) with estimated relative abundances between 0.12% and 3.62%. High rates of denitrification fueled by internally stored carbon during the anoxic phase were correlated with elevated aerobic phosphorus uptake rates. Batch tests in the latter two years indicated that anoxic phosphorus uptake rates accounted for 3% to 40% of the aerobic uptake rates, suggesting that the reduction in DO concentrations from 2019 to 2023 may have facilitated anoxic phosphorus uptake capacity.

Exposure to pathogens remains the greatest acute health concern related to potable water reuse applications. Here, we implement high-volume sample concentration for both molecular- and culture-based analyses to evaluate pathogen and surrogate removal through a 1-MGD scale coagulation, flocculation, sedimentation, ozonation, and biofiltration treatment train. The reduction of Cryptosporidium and Giardia was quantified across the wastewater treatment plant and the advanced water treatment (AWT) process. Considering the low influent concentrations, only 2 and 4 total log-reduction values (LRVs) could be demonstrated for Cryptosporidium and Giardia, respectively. Adenovirus, rotavirus, norovirus GI, and norovirus GII concentration and reduction were quantified using droplet digital polymerase chain reaction (PCR) across the AWT process. Average enteric virus LRV, through coagulation/flocculation/sedimentation, ozonation, and biofiltration, was shown to be 1.5, 0.3, and 2 LRV, respectively. Both molecular and culture-based nonpathogenic viral surrogates were shown to be representatives of enteric virus reduction by physical removal treatment processes. Due to the low concentration of indigenous pathogens and surrogates, challenge tests were performed on the pilot scale to evaluate the inactivation and removal of pathogens by ozone and biofiltration. These full-scale monitoring data and pilot challenge testing data provide validation of the pathogen LRV credit claimed in ozone-biofiltration-based AWT, which is necessary to protect public health in reuse scenarios.

Wastewater alkalinity enhancement is a promising approach for ocean alkalinity enhancement due to its potential to deliver strong bases with minimum secondary precipitation and its potential use of the global network of wastewater treatment plants (WWTPs). WWTPs are also significant sources of CO _2 due to organic matter oxidation, and integrating alkalinity addition into treatment processes may both reduce in-plant CO _2 emissions and increase downstream CO _2 uptake. This study presents a modeling framework that combines a modern activated sludge model-based WWTP simulator with an integrated hydrodynamic-biogeochemical-carbonate chemistry model of coastal oceans. We evaluate the effects of adding alkalinity either upstream (UpAdd) of the biological treatment stage or downstream at the discharge location (DnAdd) on WWTP carbon emission reduction and marine CO _2 removal. The carbon emission from WWTPs decreases with increasing alkalinity dosage in UpAdd and can be eliminated at a dosage level that is feasible to implement. However, carbon uptake in the surrounding oceanic water is much reduced due to elevated dissolved inorganic carbon in the discharge water. DnAdd does not affect CO _2 emissions from WWTPs but enhances carbon uptake in the ocean, with the net oceanic uptake of atmospheric CO _2 increasing with increasing dosage level. Across all tested dosage levels, total CO _2 removal, including emission reduction at the WWTPs and enhanced carbon uptake in the ocean, is 30% greater in UpAdd than in DnAdd . WWTP treatment tanks have much higher p CO _2 than in the ocean, and aeration of process tanks enhances the gas transfer. The upstream alkalinity addition leads to sharp declines in p CO _2 in the treatment tanks and large reductions in carbon emission from the WWTPs. These results have implications for developing strategies to reduce global carbon emission and enhance oceanic carbon burial using WWTPs as a delivery mechanism.

This paper examines the design and implementation of a full-scale integrated fixed-film activated sludge (IFAS) partial denitrification-anammox (PdNA) process at the Hampton Roads Sanitation District (HRSD) James River Treatment Plant (JRTP) in Newport News, VA, USA. The study assesses the operational outcomes of PdNA in the postanoxic zone of two full-scale demonstration treatment trains. Over 650 days, the first implementation, IFAS 1, underwent three supplemental carbon combinations for partial denitrification (PdN): step-fed primary clarifier effluent (PCE), methanol and PCE, and glycerol and PCE. Anammox bacteria were established in IFAS 1 144 days post-startup, during the second phase of carbon addition, marking the first successful full-scale deliberate application of an integrated PdNA process. Once IFAS 1's maximum anammox activity reached steady state during the final phase of carbon addition, IFAS 1 achieved an average in situ total inorganic nitrogen (TIN) removal rate of 0.93 ± 0.52 g/m2/day (0.35 ± 0.24 g/m2/day due to anammox). The second reactor, IFAS 2, was implemented a year later, using only step-fed PCE as an external carbon source. Anammox bacteria were confirmed in IFAS 2 192 days post-startup, demonstrating the potential for establishing anammox without an external carbon source and highlighting anammox bacteria's ability to establish and persist under limited nitrite (NO2 -) conditions. Although IFAS 2 demonstrated that PCE was sufficient for anammox establishment, the more limited carbon available for maintaining anoxic conditions and promoting PdN resulted in minimal in situ anammox activity and a lower maximum anammox activity (0.18 ± 0.05 g-N/m2/day). However, IFAS 1 demonstrated that supplementing external carbon with PCE can result in substantial carbon savings. At steady state, supplementing glycerol addition with PCE in IFAS 1 increased carbon savings from 43% to 75% compared to full denitrification. This study offers valuable insights into the operational dynamics, challenges, and benefits of implementing full-scale mainstream PdNA, thereby contributing to the intensification of wastewater treatment processes. PRACTITIONER POINTS: For the first time, a deliberate design and implementation of a full-scale mainstream IFAS PdNA process was successfully completed. Anammox activity was established without biomass seeding, and it was not prevented by using carbons with lower PdN efficiencies. Significant carbon cost savings were achieved by supplementing external carbon with carbon provided via step feed to the PdNA zone. Anammox's ability to remove ammonia coming with step feed to the second anoxic zone is a unique benefit of PdNA.

Aeration in biological nutrient removal (BNR) systems constitutes one of the largest energy demands in water resource recovery facilities (WRRFs). Previous studies have shown that lowering dissolved oxygen (DO) concentrations can sustain effective nitrification and phosphorus removal while substantially reducing energy consumption. However, the microbial mechanisms enabling these low-DO processes remain poorly understood. In this study, we investigated microbial communities associated with reduced DO in a pilot-scale BNR system operated by the Hampton Roads Sanitation District (HRSD). DO was reduced over an 18-month period from 2.5 mg/L to 0.2 mg/L. Metagenomic DNA was obtained from samples from each DO condition then sequenced using PacBio HiFi technology. A total of 316 metagenome-assembled genomes were recovered and after dereplication, 207 were found to be unique. These data augment the metagenomic information related to wastewater treatment under low-DO conditions and provide valuable resources for understanding microbial adaptation to oxygen-limited BNR operation. ### Competing Interest Statement The authors have declared no competing interest. United States Department of Energy, https://ror.org/01bj3aw27, DE-EE0009509, DE-SC0018409

Leveraging comammox Nitrospira and anammox bacteria for shortcut nitrogen removal can drastically lower the carbon footprint of wastewater treatment facilities by decreasing aeration energy, carbon, alkalinity, and tank volume requirements while also potentially reducing nitrous oxide emissions. However, their co-occurrence as dominant nitrifying bacteria is rarely reported in full-scale wastewater treatment. As a result, there is a poor understanding of how operational parameters, in particular, dissolved oxygen, impact their activity and synergistic behavior. Here, we report the impact of dissolved oxygen concentration (DO = 2, 4, 6 mg/L) on the microbial community's transcriptomic expression in a full-scale integrated fixed film activated sludge (IFAS) municipal wastewater treatment facility where nitrogen removal is predominantly performed by comammox Nitrospira and anammox bacterial populations. 16S rRNA transcript compositions revealed anammox bacteria and Nitrospira were significantly more active in IFAS biofilms compared to suspended sludge biomass. In IFAS biofilms, anammox bacteria significantly increased hzo expression at lower dissolved oxygen concentrations and this increase was highly correlated with the amoA expression levels of comammox bacteria. Interestingly, the genes involved in nitrite oxidation by comammox bacteria were significantly more upregulated, relative to the genes involved in ammonia oxidation with decreasing dissolved oxygen concentrations. Ultimately, our findings suggest that comammox Nitrospira supplies anammox bacteria with nitrite via ammonia oxidation and that this synergistic behavior is dependent on dissolved oxygen concentrations.