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This study presents a comprehensive analysis of the decay patterns of endogenous SARS-CoV-2 and Pepper mild mottle virus (PMMoV) within wastewaters spiked with stool from infected patients expressing COVID-19 symptoms, and hence explores the decay of endogenous SARS-CoV-2 and PMMoV targets in wastewaters from source to collection of the sample. Stool samples from infected patients were used as endogenous viral material to more accurately mirror real-world decay processes compared to more traditionally used lab-propagated spike-ins. As such, this study includes data on early decay stages of endogenous viral targets in wastewaters that are typically overlooked when performing decay studies on wastewaters harvested from wastewater treatment plants that contain already-degraded endogenous material. The two distinct sewer transport conditions of dynamic suspended sewer transport and bed and near-bed sewer transport were simulated in this study at temperatures of 4 °C, 12 °C and 20 °C to elucidate decay under these two dominant transport conditions within wastewater infrastructure. The dynamic suspended sewer transport was simulated over 35 h, representing typical flow conditions, whereas bed and near-bed transport extended to 60 days to reflect the prolonged settling of solids in sewer systems during reduced flow periods. In dynamic suspended sewer transport, no decay was observed for SARS-CoV-2, PMMoV, or total RNA over the 35-h period, and temperature ranging from 4 °C to 20 °C had no noticeable effect. Conversely, experiments simulating bed and near-bed transport conditions revealed significant decreases in SARS-CoV-2 and total RNA concentrations by day 2, and PMMoV concentrations by day 3. Only PMMoV exhibited a clear trend of increasing decay constant with higher temperatures, suggesting that while temperature influences decay dynamics, its impact may be less significant than previously assumed, particularly for endogenous RNA that is bound to dissolved organic matter in wastewater. First order decay models were inadequate for accurately fitting decay curves of SARS-CoV-2, PMMoV, and total RNA in bed and near-bed transport conditions. F-tests confirmed the superior fit of the two-phase decay model compared to first order decay models across temperatures of 4 °C-20 °C. Finally, and most importantly, total RNA normalization emerged as an appropriate approach for correcting the time decay of SARS-CoV-2 exposed to bed and near-bed transport conditions. These findings highlight the importance of considering decay from the point of entry in the sewers, sewer transport conditions, and normalization strategies when assessing and modelling the impact of viral decay rates in wastewater systems. This study also emphasizes the need for ongoing research into the diverse and multifaceted factors that influence these decay rates, which is crucial for accurate public health monitoring and response strategies., (Copyright © 2024. Published by Elsevier GmbH.)

Wastewater-based surveillance of human disease offers timely insights to public health, helping to mitigate infectious disease outbreaks and decrease downstream morbidity and mortality. These systems rely on nucleic acid amplification tests for monitoring disease trends, while antibody-based seroprevalence surveys gauge community immunity. However, serological surveys are resource-intensive and subject to potentially long lead times and sampling bias. We identified and characterized a human antibody repertoire, predominantly secretory IgA, isolated from a central wastewater treatment plant and building-scale wastewater collection points. These antibodies partition to the solids fraction and retain immunoaffinity for SARS-CoV-2 and Influenza A virus antigens. This stable pool could enable real-time tracking for correlates of vaccination, infection, and immunity, aiding in establishing population-level thresholds for immune protection and assessing the efficacy of future vaccine campaigns., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

During the COVID-19 pandemic, the monitoring of SARS-CoV-2 RNA in wastewater was used to track the evolution and emergence of variant lineages and gauge infection levels in the community, informing appropriate public health responses without relying solely on clinical testing. As more sublineages were discovered, it increased the difficulty in identifying distinct variants in a mixed population sample, particularly those without a known lineage. Here, we compare the sequencing technology from Illumina and from Oxford Nanopore Technologies, in order to determine their efficacy at detecting variants of differing abundance, using 248 wastewater samples from various Quebec and Ontario cities. Our study used two analytical approaches to identify the main variants in the samples: the presence of signature and marker mutations and the co-occurrence of signature mutations within the same amplicon. We observed that each sequencing method detected certain variants at different frequencies as each method preferentially detects mutations of distinct variants. Illumina sequencing detected more mutations with a predominant lineage that is in low abundance across the population or unknown for that time period, while Nanopore sequencing had a higher detection rate of mutations that are predominantly found in the high abundance B.1.1.7 (Alpha) lineage as well as a higher sequencing rate of co-occurring mutations in the same amplicon. We present a workflow that integrates short-read and long-read sequencing to improve the detection of SARS-CoV-2 variant lineages in mixed population samples, such as wastewater.

Background: Day-to-day variation in the measurement of SARS-CoV-2 in wastewater can challenge public health interpretation. We assessed a Bayesian smoothing and forecasting method previously used for surveillance and short-term projection of COVID-19 cases, hospitalizations, and deaths., Methods: SARS-CoV-2 viral measurement from the sewershed in Ottawa, Canada, sampled at the municipal wastewater treatment plant from July 1, 2020, to February 15, 2022, was used to assess and internally validate measurement averaging and prediction. External validation was performed using viral measurement data from influent wastewater samples from 15 wastewater treatment plants and municipalities across Ontario., Results: Plots of SARS-CoV-2 viral measurement over time using Bayesian smoothing visually represented distinct COVID-19 "waves" described by case and hospitalization data in both initial (Ottawa) and external validation in 15 Ontario communities. The time-varying growth rate of viral measurement in wastewater samples approximated the growth rate observed for cases and hospitalization. One-week predicted viral measurement approximated the observed viral measurement throughout the assessment period from December 23, 2020, to August 8, 2022. An uncalibrated model showed underprediction during rapid increases in viral measurement (positive growth) and overprediction during rapid decreases. After recalibration, the model showed a close approximation between observed and predicted estimates., Conclusion: Bayesian smoothing of wastewater surveillance data of SARS-CoV-2 allows for accurate estimates of COVID-19 growth rates and one- and two-week forecasting of SARS-CoV-2 in wastewater for 16 municipalities in Ontario, Canada. Further assessment is warranted in other communities representing different sewersheds and environmental conditions., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario., (© 2024. The Author(s).)

Wastewater surveillance of coronavirus disease 2019 (COVID-19) commonly applies reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to quantify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater over time. In most applications worldwide, maximal sensitivity and specificity of RT-qPCR has been achieved, in part, by monitoring two or more genomic loci of SARS-CoV-2. In Ontario, Canada, the provincial Wastewater Surveillance Initiative reports the average copies of the CDC N1 and N2 loci normalized to the fecal biomarker pepper mild mottle virus. In November 2021, the emergence of the Omicron variant of concern, harboring a C28311T mutation within the CDC N1 probe region, challenged the accuracy of the consensus between the RT-qPCR measurements of the N1 and N2 loci of SARS-CoV-2. In this study, we developed and applied a novel real-time dual loci quality assurance and control framework based on the relative difference between the loci measurements to the City of Ottawa dataset to identify a loss of sensitivity of the N1 assay in the period from July 10, 2022 to January 31, 2023. Further analysis via sequencing and allele-specific RT-qPCR revealed a high proportion of mutations C28312T and A28330G during the study period, both in the City of Ottawa and across the province. It is hypothesized that nucleotide mutations in the probe region, especially A28330G, led to inefficient annealing, resulting in reduction in sensitivity and accuracy of the N1 assay. This study highlights the importance of implementing quality assurance and control criteria to continually evaluate, in near real-time, the accuracy of the signal produced in wastewater surveillance applications that rely on detection of pathogens whose genomes undergo high rates of mutation., (© 2024. The Author(s).)

Respiratory syncytial virus (RSV) is the leading viral cause of childhood bronchiolitis and pneumonia causing over 3 million hospitalizations and 100,000 deaths in children under 5 years of age annually. Wastewater-based surveillance (WBS) has proven an effective early warning system for high-consequence pathogens, including SARS-CoV-2, polio, mpox, and influenza, but has yet to be fully leveraged for RSV surveillance. A model predicated on the Canadian province of Ontario demonstrates that implementation of a WBS system can potentially result in significant cost savings and clinical benefits when guiding an RSV preventive program with a long-acting monoclonal antibody. A network of integrated WBS initiatives offers the opportunity to help minimize the devastating global burden of RSV in children by optimizing the timing of preventive measures and we strongly advocate that its benefits continue to be explored., Competing Interests: RD and EM have no conflicts to declare regarding this study but hold leadership positions in a company (Advanced Environmental Molecular Analytics Ltd.) with work non-relevant to the work presented. BP has received research funding and/or compensation as advisor/lecturer from AstraZeneca and Sanofi outside the scope of this study. BR-G and JE were employed by the company Violicom Medical Limited. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Thampi, Mercier, Paes, Edwards, Rodgers-Gray and Delatolla.)

Wastewater surveillance (WWS) of SARS-CoV-2 has become a crucial tool for monitoring COVID-19 cases and outbreaks. Previous studies have indicated that SARS-CoV-2 RNA measurement from testing solid-rich primary sludge yields better sensitivity compared to testing wastewater influent. Furthermore, measurement of pepper mild mottle virus (PMMoV) signal in wastewater allows for precise normalization of SARS-CoV-2 viral signal based on solid content, enhancing disease prevalence tracking. However, despite the widespread adoption of WWS, a knowledge gap remains regarding the impact of ferric sulfate coagulation, commonly used in enhanced primary clarification, the initial stage of wastewater treatment where solids are sedimented and removed, on SARS-CoV-2 and PMMoV quantification in wastewater-based epidemiology. This study examines the effects of ferric sulfate addition, along with the associated pH reduction, on the measurement of SARS-CoV-2 and PMMoV viral measurements in wastewater primary clarified sludge through jar testing. Results show that the addition of Fe 3+ concentrations in the conventional 0 to 60 mg/L range caused no effect on SARS-CoV-2 N1 and N2 gene region measurements in wastewater solids. However, elevated Fe 3+ concentrations were shown to be associated with a statistically significant increase in PMMoV viral measurements in wastewater solids, which consequently resulted in the underestimation of PMMoV-normalized SARS-CoV-2 viral signal measurements (N1 and N2 copies/copies of PMMoV). The observed pH reduction from coagulant addition did not contribute to the increased PMMoV measurements, suggesting that this phenomenon arises from the partitioning of PMMoV viral particles into wastewater solids., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Importance: Cheese production facilities must abide by sewage discharge bylaws that prevent overloading municipal water resource recovery facilities, eutrophication, and toxicity to aquatic life. Compact treatment systems can permit on-site treatment of cheese production wastewater; however, competition between heterotrophs and nitrifiers impedes the implementation of the sequencing batch moving bed biofilm reactor (SB-MBBR) for nitrification from high-carbon wastewaters. This study demonstrates that a single SB-MBBR is not feasible for nitrification when operated with anerobic and aerobic cycling for carbon and phosphorous removal from cheese production wastewater, as nitrification does not occur in a single reactor. Thus, two reactors in series are recommended to achieve nitrification from cheese production wastewater in SB-MBBRs. These findings can be applied to pilot and full-scale SB-MBBR operations. By demonstrating the potential to implement partial nitrification in the SB-MBBR system, this study presents the possibility of implementing partial nitrification in the SB-MBBR, resulting in the potential for more sustainable treatment of nitrogen from cheese production wastewater., Competing Interests: The authors declare no conflict of interest.

Wastewater-based surveillance is a valuable approach for monitoring COVID-19 at community level. Monitoring SARS-CoV-2 variants of concern (VOC) in wastewater has become increasingly relevant when clinical testing capacity and case-based surveillance are limited. In this study, we ascertained the turnover of six VOC in Alberta wastewater from May 2020 to May 2022. Wastewater samples from nine wastewater treatment plants across Alberta were analysed using VOC-specific RT-qPCR assays. The performance of the RT-qPCR assays in identifying VOC in wastewater was evaluated against next generation sequencing. The relative abundance of each VOC in wastewater was compared to positivity rate in COVID-19 testing. VOC-specific RT-qPCR assays performed comparatively well against next generation sequencing; concordance rates ranged from 89% to 98% for detection of Alpha, Beta, Gamma, Omicron BA.1 and Omicron BA.2, with a slightly lower rate of 85% for Delta ( p <  0.01). Elevated relative abundance of Alpha, Delta, Omicron BA.1 and BA.2 were each associated with increased COVID-19 positivity rate. Alpha, Delta and Omicron BA.2 reached 90% relative abundance in wastewater within 80, 111 and 62 days after their initial detection, respectively. Omicron BA.1 increased more rapidly, reaching a 90% relative abundance in wastewater after 35 days. Our results from VOC surveillance in wastewater correspond with clinical observations that Omicron is the VOC with highest disease burden over the shortest period in Alberta to date. The findings suggest that changes in relative abundance of a VOC in wastewater can be used as a supplementary indicator to track and perhaps predict COVID-19 burden in a population.

Introduction: Detection of community respiratory syncytial virus (RSV) infections informs the timing of immunoprophylaxis programs and hospital preparedness for surging pediatric volumes. In many jurisdictions, this relies upon RSV clinical test positivity and hospitalization (RSVH) trends, which are lagging indicators. Wastewater-based surveillance (WBS) may be a novel strategy to accurately identify the start of the RSV season and guide immunoprophylaxis administration and hospital preparedness., Methods: We compared citywide wastewater samples and pediatric RSVH in Ottawa and Hamilton between August 1, 2022, and March 5, 2023. 24-h composite wastewater samples were collected daily and 5 days a week at the wastewater treatment facilities in Ottawa and Hamilton, Ontario, Canada, respectively. RSV WBS samples were analyzed in real-time for RSV by RT-qPCR., Results: RSV WBS measurements in both Ottawa and Hamilton showed a lead time of 12 days when comparing the WBS data set to pediatric RSVH data set (Spearman's ρ = 0.90). WBS identify early RSV community transmission and declared the start of the RSV season 36 and 12 days in advance of the provincial RSV season start (October 31) for the city of Ottawa and Hamilton, respectively. The differing RSV start dates in the two cities is likely associated with geographical and regional variation in the incidence of RSV between the cities., Discussion: Quantifying RSV in municipal wastewater forecasted a 12-day lead time of the pediatric RSVH surge and an earlier season start date compared to the provincial start date. These findings suggest an important role for RSV WBS to inform regional health system preparedness, reduce RSV burden, and understand variations in community-related illness as novel RSV vaccines and monoclonal antibodies become available., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mercier, Pisharody, Guy, Wan, Hegazy, D’Aoust, Kabir, Nguyen, Eid, Harvey, Rodenburg, Rutherford, Mackenzie, Willmore, Hui, Paes, Delatolla and Thampi.)

Monitoring of viral signal in wastewater is considered a useful tool for monitoring the burden of COVID-19, especially during times of limited availability in testing. Studies have shown that COVID-19 hospitalizations are highly correlated with wastewater viral signals and the increases in wastewater viral signals can provide an early warning for increasing hospital admissions. The association is likely nonlinear and time-varying. This project employs a distributed lag nonlinear model (DLNM) (Gasparrini et al., 2010) to study the nonlinear exposure-response delayed association of the COVID-19 hospitalizations and SARS-CoV-2 wastewater viral signals using relevant data from Ottawa, Canada. We consider up to a 15-day time lag from the average of SARS-CoV N1 and N2 gene concentrations to COVID-19 hospitalizations. The expected reduction in hospitalization is adjusted for vaccination efforts. A correlation analysis of the data verifies that COVID-19 hospitalizations are highly correlated with wastewater viral signals with a time-varying relationship. Our DLNM based analysis yields a reasonable estimate of COVID-19 hospitalizations and enhances our understanding of the association of COVID-19 hospitalizations with wastewater viral signals., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Recent MPOX viral resurgences have mobilized public health agencies around the world. Recognizing the significant risk of MPOX outbreaks, large-scale human testing, and immunization campaigns have been initiated by local, national, and global public health authorities. Recently, traditional clinical surveillance campaigns for MPOX have been complemented with wastewater surveillance (WWS), building on the effectiveness of existing wastewater programs that were built to monitor SARS-CoV-2 and recently expanded to include influenza and respiratory syncytial virus surveillance in wastewaters. In the present study, we demonstrate and further support the finding that MPOX viral fragments agglomerate in the wastewater solids fraction. Furthermore, this study demonstrates that the current, most commonly used MPOX assays are equally effective at detecting low titers of MPOX viral signal in wastewaters. Finally, MPOX WWS is shown to be more effective at passively tracking outbreaks and/or resurgences of the disease than clinical testing alone in smaller communities with low human clinical case counts of MPOX.

Introduction: Wastewater-based surveillance is at the forefront of monitoring for community prevalence of COVID-19, however, continued uncertainty exists regarding the use of fecal indicators for normalization of the SARS-CoV-2 virus in wastewater. Using three communities in Ontario, sampled from 2021-2023, the seasonality of a viral fecal indicator (pepper mild mottle virus, PMMoV) and the utility of normalization of data to improve correlations with clinical cases was examined., Methods: Wastewater samples from Warden, the Humber Air Management Facility (AMF), and Kitchener were analyzed for SARS-CoV-2, PMMoV, and crAssphage. The seasonality of PMMoV and flow rates were examined and compared by Season-Trend-Loess decomposition analysis. The effects of normalization using PMMoV, crAssphage, and flow rates were analyzed by comparing the correlations to clinical cases by episode date (CBED) during 2021., Results: Seasonal analysis demonstrated that PMMoV had similar trends at Humber AMF and Kitchener with peaks in January and April 2022 and low concentrations (troughs) in the summer months. Warden had similar trends but was more sporadic between the peaks and troughs for PMMoV concentrations. Flow demonstrated similar trends but was not correlated to PMMoV concentrations at Humber AMF and was very weak at Kitchener ( r  = 0.12). Despite the differences among the sewersheds, unnormalized SARS-CoV-2 (raw N1-N2) concentration in wastewater ( n  = 99-191) was strongly correlated to the CBED in the communities ( r  = 0.620-0.854) during 2021. Additionally, normalization with PMMoV did not improve the correlations at Warden and significantly reduced the correlations at Humber AMF and Kitchener. Flow normalization ( n  = 99-191) at Humber AMF and Kitchener and crAssphage normalization ( n  = 29-57) correlations at all three sites were not significantly different from raw N1-N2 correlations with CBED., Discussion: Differences in seasonal trends in viral biomarkers caused by differences in sewershed characteristics (flow, input, etc.) may play a role in determining how effective normalization may be for improving correlations (or not). This study highlights the importance of assessing the influence of viral fecal indicators on normalized SARS-CoV-2 or other viruses of concern. Fecal indicators used to normalize the target of interest may help or hinder establishing trends with clinical outcomes of interest in wastewater-based surveillance and needs to be considered carefully across seasons and sites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Dhiyebi, Abu Farah, Ikert, Srikanthan, Hayat, Bragg, Qasim, Payne, Kaleis, Paget, Celmer-Repin, Folkema, Drew, Delatolla, Giesy and Servos.)

We determined correlations between SARS-CoV-2 load in untreated water and COVID-19 cases and patient hospitalizations before the Omicron variant (September 2020-November 2021) at 2 wastewater treatment plants in the Regional Municipality of Peel, Ontario, Canada. Using pre-Omicron correlations, we estimated incident COVID-19 cases during Omicron outbreaks (November 2021-June 2022). The strongest correlation between wastewater SARS-CoV-2 load and COVID-19 cases occurred 1 day after sampling (r = 0.911). The strongest correlation between wastewater load and COVID-19 patient hospitalizations occurred 4 days after sampling (r = 0.819). At the peak of the Omicron BA.2 outbreak in April 2022, reported COVID-19 cases were underestimated 19-fold because of changes in clinical testing. Wastewater data provided information for local decision-making and are a useful component of COVID-19 surveillance systems.

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mark Servos reports financial support was provided by Ontario Ministry of the Environment Conservation and Parks. Mark Servos reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Mark Servos reports financial support was provided by Global Water Futures., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) has been demonstrated for its great potential in tracking of coronavirus disease 2019 (COVID-19) transmission among populations despite some inherent methodological limitations. These include non-optimized sampling approaches and analytical methods; stability of viruses in sewer systems; partitioning/retention in biofilms; and the singular and inaccurate back-calculation step to predict the number of infected individuals in the community. Future research is expected to (1) standardize best practices in wastewater sampling, analysis and data reporting protocols for the sensitive and reproducible detection of viruses in wastewater; (2) understand the in-sewer viral stability and partitioning under the impacts of dynamic wastewater flow, properties, chemicals, biofilms and sediments; and (3) achieve smart wastewater surveillance with artificial intelligence and big data models. Further specific research is essential in the monitoring of other viral pathogens with pandemic potential and subcatchment applications to maximize the benefits of WBE beyond COVID-19., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Guangming Jiang reports financial support was provided by Australian Research Council. Yanchen Liu reports financial support was provided by National Natural Science Foundation of China. Yanchen Liu reports financial support was provided by Tsinghua University. Guangming Jiang reports financial support was provided by Australian Academy of Science., (© 2023 Elsevier B.V. All rights reserved.)

There is a current need for a low operational intensity, effective and small footprint system to achieve stable partial nitritation for subsequent anammox treatment at mainstream municipal wastewaters. This research identifies a unique design strategy using an elevated total ammonia nitrogen (TAN) surface area loading rate (SALR) of 5 g TAN/m 2. d to achieve cost-effective, stable, and elevated rates of partial nitritation in a moving bed biofilm reactor (MBBR) system under mainstream conditions. The elevated loaded partial nitritation MBBR system achieves a TAN surface area removal rate (SARR) of 2.01 ± 0.07 g TAN/m 2. d and NO 2 - -N: NH 4 + -N stoichiometric ratio of 1.15:1, which is appropriate for downstream anammox treatment. The elevated TAN SALR design strategy promotes nitrite-oxidizing bacteria (NOB) activity suppression rather than a reduction in NOB population as the reason for the suppression of nitrite oxidation in the mainstream elevated loaded partial nitritation MBBR system. NOB activity is limited at an elevated TAN SALR likely due to thick biofilm embedding the NOB population and competition for dissolved oxygen (DO) with ammonia-oxidizing bacteria for TAN oxidation to nitrite within the biofilm structure, which ultimately limits the uptake of DO by NOB in the system. Therefore, this design strategy offers a cost-effective and efficient alternative for mainstream partial nitritation MBBR systems at water resource recovery facilities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ikem, Chen and Delatolla.)

The real-time polymerase chain reaction (PCR), commonly known as quantitative PCR (qPCR), is increasingly common in environmental microbiology applications. During the COVID-19 pandemic, qPCR combined with reverse transcription (RT-qPCR) has been used to detect and quantify SARS-CoV-2 in clinical diagnoses and wastewater monitoring of local trends. Estimation of concentrations using qPCR often features a log-linear standard curve model calibrating quantification cycle ( Cq ) values obtained from underlying fluorescence measurements to standard concentrations. This process works well at high concentrations within a linear dynamic range but has diminishing reliability at low concentrations because it cannot explain "non-standard" data such as Cq values reflecting increasing variability at low concentrations or non-detects that do not yield Cq values at all. Here, fundamental probabilistic modeling concepts from classical quantitative microbiology were integrated into standard curve modeling approaches by reflecting well-understood mechanisms for random error in microbial data. This work showed that data diverging from the log-linear regression model at low concentrations as well as non-detects can be seamlessly integrated into enhanced standard curve analysis. The newly developed model provides improved representation of standard curve data at low concentrations while converging asymptotically upon conventional log-linear regression at high concentrations and adding no fitting parameters. Such modeling facilitates exploration of the effects of various random error mechanisms in experiments generating standard curve data, enables quantification of uncertainty in standard curve parameters, and is an important step toward quantifying uncertainty in qPCR-based concentration estimates. Improving understanding of the random error in qPCR data and standard curve modeling is especially important when low concentrations are of particular interest and inappropriate analysis can unduly affect interpretation, conclusions regarding lab performance, reported concentration estimates, and associated decision-making., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schmidt, Acosta, Chik, D’Aoust, Delatolla, Dhiyebi, Glier, Hubert, Kopetzky, Mangat, Pang, Peterson, Prystajecky, Qiu, Servos and Emelko.)

Wastewater surveillance (WWS) of SARS-CoV-2 was proven to be a reliable and complementary tool for population-wide monitoring of COVID-19 disease incidence but was not as rigorously explored as an indicator for disease burden throughout the pandemic. Prior to global mass immunization campaigns and during the spread of the wildtype COVID-19 and the Alpha variant of concern (VOC), viral measurement of SARS-CoV-2 in wastewater was a leading indicator for both COVID-19 incidence and disease burden in communities. As the two-dose vaccination rates escalated during the spread of the Delta VOC in Jul. 2021 through Dec. 2021, relations weakened between wastewater signal and community COVID-19 disease incidence and maintained a strong relationship with clinical metrics indicative of disease burden (new hospital admissions, ICU admissions, and deaths). Further, with the onset of the vaccine-resistant Omicron BA.1 VOC in Dec. 2021 through Mar. 2022, wastewater again became a strong indicator of both disease incidence and burden during a period of limited natural immunization (no recent infection), vaccine escape, and waned vaccine effectiveness. Lastly, with the populations regaining enhanced natural and vaccination immunization shortly prior to the onset of the Omicron BA.2 VOC in mid-Mar 2022, wastewater is shown to be a strong indicator for both disease incidence and burden. Hospitalization-to-wastewater ratio is further shown to be a good indicator of VOC virulence when widespread clinical testing is limited. In the future, WWS is expected to show moderate indication of incidence and strong indication of disease burden in the community during future potential seasonal vaccination campaigns., Competing Interests: Declaration of competing interest The authors declare that no competing financial interests or personal relationships influenced the work reported in this manuscript., (Copyright © 2022. Published by Elsevier B.V.)

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) will have greater value as an important diagnostic tool. An in-depth analysis and understanding of the metrics derived from WWS is required to interpret and utilize WWS-acquired data effectively (McClary-Gutierrez et al., 2021; O'Keeffe, 2021). In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven cities in Canada over periods ranging from 8 to 21 months. This work demonstrates that significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing (resulting in a reduction to testing access and a reduction in the number of daily tests) in these communities, despite increases in the wastewater signal. Furthermore, the WC ratio decreased significantly in 6 of the 7 studied locations, serving as a potential signal of the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community (40-60 % allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized community (40-60 % allelic proportion). Finally, a significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variant's greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when community immunity was high. The WC ratio, used as an additional monitoring metric, could complement clinical case counts and wastewater signals as individual metrics in its potential ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics., Competing Interests: Declaration of competing interest The authors declare that no known competing financial interests or personal relationships influenced the work reported in this manuscript., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The relatively poor settling characteristics of particles produced in moving bed biofilm reactor (MBBR) outline the importance of developing a fundamental understanding of the characterization and settleability of MBBR-produced solids. The influence of carrier geometric properties and different levels of biofilm thickness on biofilm characteristics, solids production, particle size distribution (PSD), and particle settling velocity distribution (PSVD) is evaluated in this study. The analytical ViCAs method is applied to the MBBR effluent to assess the distribution of particle settling velocities. This method is combined with microscopy imaging to relate particle size distribution to settling velocity. Three conventionally loaded MBBR systems are studied at a similar loading rate of 6.0 g/(m 2  •day) and with different carrier types. The AnoxK™ K5 carrier, a commonly used carrier, is compared to so-called thickness-restraint carriers, AnoxK™ Z-carriers that are newly designed carriers to limit the biofilm thickness. Moreover, two levels of biofilm thickness, 200 μm and 400 μm, are studied using AnoxK™ Z-200 and Z-400 carriers. Statistical analysis confirms that K5 carriers demonstrated a significantly different biofilm mass, thickness, and density, in addition to distinct trends in PSD and PSVD in comparison with Z-carriers. However, in comparison of thickness-restraint carriers, Z-200 carrier results did not vary significantly compared to the Z-400 carrier. The K5 carriers showed the lowest production of suspended solids (0.7 ± 0.3 g-TSS/day), thickest biofilm (281.1 ± 8.7 µm) and lowest biofilm density (65.0 ± 1.5 kg/m 3 ). The K5 effluent solids also showed enhanced settling behaviour, consisting of larger particles with faster settling velocities., (Copyright © 2021. Published by Elsevier B.V.)

Recurrent influenza epidemics and pandemic potential are significant risks to global health. Public health authorities use clinical surveillance to locate and monitor influenza and influenza-like cases and outbreaks to mitigate hospitalizations and deaths. Currently, global integration of clinical surveillance is the only reliable method for reporting influenza types and subtypes to warn of emergent pandemic strains. The utility of wastewater surveillance (WWS) during the COVID-19 pandemic as a less resource intensive replacement or complement for clinical surveillance has been predicated on analyzing viral fragments in wastewater. We show here that influenza virus targets are stable in wastewater and partitions favorably to the solids fraction. By quantifying, typing, and subtyping the virus in municipal wastewater and primary sludge during a community outbreak, we forecasted a citywide flu outbreak with a 17-day lead time and provided population-level viral subtyping in near real-time to show the feasibility of influenza virus WWS at the municipal and neighbourhood levels in near real time using minimal resources and infrastructure., (© 2022. The Author(s).)

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.

Amid the 2019 coronavirus disease pandemic (COVID-19), the scientific community has a responsibility to provide accessible public health resources within their communities. Wastewater based epidemiology (WBE) has been used to monitor community spread of the pandemic. The goal of this review was to evaluate the need for an environmental justice approach for COVID-19 WBE starting with the state of California in the United States. Methods included a review of the peer-reviewed literature, government-provided data, and news stories. As of June 2021, there were twelve universities, nine public dashboards, and 48 of 384 wastewater treatment plants monitoring wastewater for SARS-CoV-2 within California. The majority of wastewater monitoring in California has been conducted in the urban areas of Coastal and Southern California (34/48), with a lack of monitoring in more rural areas of Central (10/48) and Northern California (4/48). Similar to the access to COVID-19 clinical testing and vaccinations, there is a disparity in access to wastewater testing which can often provide an early warning system to outbreaks. This research demonstrates the need for an environmental justice approach and equity considerations when determining locations for environmental monitoring., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (© 2022 The Author(s).)

The COVID-19 pandemic has stimulated wastewater-based surveillance, allowing public health to track the epidemic by monitoring the concentration of the genetic fingerprints of SARS-CoV-2 shed in wastewater by infected individuals. Wastewater-based surveillance for COVID-19 is still in its infancy. In particular, the quantitative link between clinical cases observed through traditional surveillance and the signals from viral concentrations in wastewater is still developing and hampers interpretation of the data and actionable public-health decisions. We present a modelling framework that includes both SARS-CoV-2 transmission at the population level and the fate of SARS-CoV-2 RNA particles in the sewage system after faecal shedding by infected persons in the population. Using our mechanistic representation of the combined clinical/wastewater system, we perform exploratory simulations to quantify the effect of surveillance effectiveness, public-health interventions and vaccination on the discordance between clinical and wastewater signals. We also apply our model to surveillance data from three Canadian cities to provide wastewater-informed estimates for the actual prevalence, the effective reproduction number and incidence forecasts. We find that wastewater-based surveillance, paired with this model, can complement clinical surveillance by supporting the estimation of key epidemiological metrics and hence better triangulate the state of an epidemic using this alternative data source., (Crown Copyright © 2022. Published by Elsevier B.V. All rights reserved.)

This study investigates and compares the ammonia removal kinetics, attachment, biofilm development and anammox bacteria enrichment on various surface modified carriers throughout the 163 days of start-up of an MBBR system: virgin, dextran-functionalized carriers, silica-functionalized and pre-seeded denitrifying carriers. Silica-functionalized carriers along with pre-seeded denitrifying carriers induced significant higher kinetics, faster biofilm growth and greater anammox bacteria enrichment during the 64 days of operation compared to non-modified virgin and dextran-functionalized carriers. The elevated anammox bacteria counts along with the elevated kinetics of all carriers measured at day 106 indicated that the completed biofilm growth and biofilm maturation are achieved prior to or at day 106 of start-up. The NH 4 + -N removal rate for virgin, dextran-functionalized, silica-functionalized and pre-seeded denitrifying carriers were achieved 0.684 ± 0.019, 0.608 ± 0.016, 0.634 ± 0.017 and 0.665 ± 0.018 g NH 4 + -N/m 2 /d, respectively, at day 106. The results demonstrate that the silica-functionalized and pre-seeded denitrifying carriers offer advantages during the early stage of start-up while the dextran-functionalized carriers did not reduce the start-up period for anammox biofilm., Competing Interests: Declaration of competing interest The authors declare that no known competing financial interests or personal relationships could appear to influence the work reported in this manuscript., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology/wastewater surveillance has been a topic of significant interest over the last year due to its application in SARS-CoV-2 surveillance to track prevalence of COVID-19 in communities. Although SARS-CoV-2 surveillance has been applied in more than 50 countries to date, the application of this surveillance has been largely focused on relatively affluent urban and peri-urban communities. As such, there is a knowledge gap regarding the implementation of reliable wastewater surveillance in small and rural communities for the purpose of tracking rates of incidence of COVID-19 and other pathogens or biomarkers. This study examines the relationships existing between SARS-CoV-2 viral signal from wastewater samples harvested from an upstream pumping station and from an access port at a downstream wastewater treatment lagoon with the community's COVID-19 rate of incidence (measured as percent test positivity) in a small, rural community in Canada. Real-time quantitative polymerase chain reaction (RT-qPCR) targeting the N1 and N2 genes of SARS-CoV-2 demonstrate that all 24-h composite samples harvested from the pumping station over a period of 5.5 weeks had strong viral signal, while all samples 24-h composite samples harvested from the lagoon over the same period were below the limit of quantification. RNA concentrations and integrity of samples harvested from the lagoon were both lower and more variable than from samples from the upstream pumping station collected on the same date, indicating a higher overall stability of SARS-CoV-2 RNA upstream of the lagoon. Additionally, measurements of PMMoV signal in wastewater allowed normalizing SARS-CoV-2 viral signal for fecal matter content, permitting the detection of actual changes in community prevalence with a high level of granularity. As a result, in sewered small and rural communities or low-income regions operating wastewater lagoons, samples for wastewater surveillance should be harvested from pumping stations or the sewershed as opposed to lagoons., Competing Interests: Declaration of competing interest The authors declare that no known competing financial interests or personal relationships could appear to influence the work reported in this manuscript., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed millions of lives to date. Antigenic drift has resulted in viral variants with putatively greater transmissibility, virulence, or both. Early and near real-time detection of these variants of concern (VOC) and the ability to accurately follow their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which uses nucleic acid amplification tests to detect viral fragments, is a reliable proxy of COVID-19 incidence and prevalence, and thus offers the potential to monitor VOC viral load in a given population. Here, we describe and validate a primer extension PCR strategy targeting a signature mutation in the N gene of SARS-CoV-2. This allows quantification of B.1.1.7 versus non-B.1.1.7 allele frequency in wastewater without the need to employ quantitative RT-PCR standard curves. We show that the wastewater B.1.1.7 profile correlates with its clinical counterpart and benefits from a near real-time and facile data collection and reporting pipeline. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost; allowing early and contemporaneous estimates of B.1.1.7 community transmission prior to, or in lieu of, clinical screening and identification. Our study demonstrates that this strategy can provide public health units with an additional and much needed tool to rapidly triangulate VOC incidence/prevalence with high sensitivity and lineage specificity., (Copyright © 2021. Published by Elsevier Ltd.)

Detection of SARS-CoV-2 RNA in wastewater is a promising tool for informing public health decisions during the COVID-19 pandemic. However, approaches for its analysis by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) are still far from standardized globally. To characterize inter- and intra-laboratory variability among results when using various methods deployed across Canada, aliquots from a real wastewater sample were spiked with surrogates of SARS-CoV-2 (gamma-radiation inactivated SARS-CoV-2 and human coronavirus strain 229E [HCoV-229E]) at low and high levels then provided "blind" to eight laboratories. Concentration estimates reported by individual laboratories were consistently within a 1.0-log 10 range for aliquots of the same spiked condition. All laboratories distinguished between low- and high-spikes for both surrogates. As expected, greater variability was observed in the results amongst laboratories than within individual laboratories, but SARS-CoV-2 RNA concentration estimates for each spiked condition remained mostly within 1.0-log 10 ranges. The no-spike wastewater aliquots provided yielded non-detects or trace levels (<20 gene copies/mL) of SARS-CoV-2 RNA. Detections appear linked to methods that included or focused on the solids fraction of the wastewater matrix and might represent in-situ SARS-CoV-2 to the wastewater sample. HCoV-229E RNA was not detected in the no-spike aliquots. Overall, all methods yielded comparable results at the conditions tested. Partitioning behavior of SARS-CoV-2 and spiked surrogates in wastewater should be considered to evaluate method effectiveness. A consistent method and laboratory to explore wastewater SARS-CoV-2 temporal trends for a given system, with appropriate quality control protocols and documented in adequate detail should succeed., (Copyright © 2021. Published by Elsevier B.V.)

Curtailing the Spring 2020 COVID-19 surge required sweeping and stringent interventions by governments across the world. Wastewater-based COVID-19 epidemiology programs have been initiated in many countries to provide public health agencies with a complementary disease tracking metric and non-discriminating surveillance tool. However, their efficacy in prospectively capturing resurgences following a period of low prevalence is unclear. In this study, the SARS-CoV-2 viral signal was measured in primary clarified sludge harvested every two days at the City of Ottawa's water resource recovery facility during the summer of 2020, when clinical testing recorded daily percent positivity below 1%. In late July, increases of >400% in normalized SARS-CoV-2 RNA signal in wastewater were identified 48 h prior to reported >300% increases in positive cases that were retrospectively attributed to community-acquired infections. During this resurgence period, SARS-CoV-2 RNA signal in wastewater preceded the reported >160% increase in community hospitalizations by approximately 96 h. This study supports wastewater-based COVID-19 surveillance of populations in augmenting the efficacy of diagnostic testing, which can suffer from sampling biases or timely reporting as in the case of hospitalization census., Competing Interests: Declaration of competing interest The authors declare that no known competing financial interests or personal relationships could appear to influence the work reported in this manuscript., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Competing Interests: None declared.

In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track community infections to inform public health interventions aimed at reducing the spread and therefore reduce pressures on health-care, improve health outcomes and reduce economic uncertainty. Wastewater surveillance has rapidly emerged as a potential tool to effectively monitor community infections through measuring trends of RNA signal in wastewater systems. In this study SARS-CoV-2 viral RNA N1 and N2 gene regions are quantified in solids collected from influent post grit solids (PGS) and primary clarified sludge (PCS) in two water resource recovery facilities (WRRF) serving Canada's national capital region, i.e., the City of Ottawa, ON (pop. ≈ 1.1M) and the City of Gatineau, QC (pop. ≈ 280K). PCS samples show signal inhibition using RT-ddPCR compared to RT-qPCR, with PGS samples showing similar quantifiable concentrations of RNA using both assays. RT-qPCR shows higher frequency of detection of N1 and N2 gene regions in PCS (92.7, 90.6%, n = 6) as compared to PGS samples (79.2, 82.3%, n = 5). Sampling of PCS may therefore be an effective approach for SARS-CoV-2 viral quantification, especially during periods of declining and low COVID-19 incidence in the community. The pepper mild mottle virus (PMMoV) is determined to have a less variable RNA signal in PCS over a three month period for two WRRFs, regardless of environmental conditions, compared to Bacteroides 16S rRNA or human 18S rRNA, making PMMoV a potentially useful biomarker for normalization of SARS-CoV-2 signal. PMMoV-normalized PCS RNA signal from WRRFs of two cities correlated with the regional public health epidemiological metrics, identifying PCS normalized to a fecal indicator (PMMoV) as a potentially effective tool for monitoring trends during decreasing and low-incidence of infection of SARS-Cov-2 in communities., Competing Interests: Declaration of Competing Interest The authors declare that no known competing financial interests or personal relationships could appear to influence the work reported in this manuscript., (Copyright © 2020. Published by Elsevier Ltd.)

This study investigates the kinetics, attachment, biofilm development and anammox bacteria enrichment of a novel detached anammox biofilm inoculation method on non-modified virgin MBBR carriers and pre-seeded denitrifying carriers. The study compares these results to the more common use of attached anammox carriers for anammox MBBR inoculation. The anammox bacteria specific attachment-growth rates for virgin carriers inoculated with detached anammox biofilm mass were 38.1% greater for the first 25 days, leading to approximately 30% less time required to achieve complete biofilm coverage than those measured in attached biofilm carrier inoculated systems during the attachment and early biofilm growth stages. The biofilm thickness increase rate was also 52.3% higher for virgin carriers with detached biofilm inoculum. Further, inoculation using pre-seeded denitrifying carriers compared to virgin carriers demonstrated a 13.8% preferential increase in anammox bacteria specific attachment-growth rate and a corresponding 47.2% higher NH 4 + -N removal rate at the time of biofilm maturation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Measurement and prediction of wastewater quality parameters are crucial for evaluating the risk to the receiving waters. This study presents new methods for the identification of outlier data and smoothing as an effective pre-processing technique prito to modelling. This new data processing method uses a combination of the autoregressive integrated moving average (ARIMA) model and -the adaptive neuro fuzzy inference system with fuzzy C-means clustering (FCM) (ANFIS-FCM). These new pre-processing methodsare compared to previously employed non-linear approaches for modelling of wastewater influent/effluent 5-day biochemical oxygen demand (BOD 5 ), chemical oxygen demand (COD) and total suspended solids (TSS). Linear modelling of each parameter, 242 linear models, were investigated, and a linear model for each parameter was selected. The results of the non-linear models led to an acceptable prediction for qualitative parameters so that the high coefficient of determination (R 2 ) was observed for the for all models was recorded as 0.8-0.87 and 0.83-0.89, respectively. By a combination of the linear and non-linear mothods a hybrid model was introduced. The proposed hybrid model for the influent BOD with the highest correlation between the observed and predicted values, and limited scattering was identified as the optimal model (R BOD and TSS , respectively. The range of the R 2 for all models was recorded as 0.8-0.87 and 0.83-0.89, respectively. By a combination of the linear and non-linear mothods a hybrid model was introduced. The proposed hybrid model for the influent BOD with the highest correlation between the observed and predicted values, and limited scattering was identified as the optimal model (R 2  = 0.95). The use of hybrid models to predict wastewater quality parameters improved the performance and efficiency of the models. In addition, a comparison of the hybrid model with the recently developed models in the literature indicates that the developed ARIMA-ANFIS-FCM outperformed other models., Competing Interests: Conflict of interest declarationThe authors declare no conflict of interest., (© Springer Nature Switzerland AG 2020.)

The food and beverage industry produces wastewaters containing high concentrations of organic carbon and nutrients, which when discharged leads to eutrophication and algal blooms. Given recent stringencies in effluent regulations, industries are required to treat their wastewater on-site. There is a critical need for compact, high-rate, cost-effective wastewater technologies to treat industrial wastewaters, such as the sequencing batch moving bed biofilm reactor. The aim of this study is to investigate the potential and evaluate the performance of the sequencing batch moving bed biofilm reactor cycling between anaerobic and aerobic stages to treat high-strength food and beverage wastewaters. Specifically, this study focuses on the effects of anaerobic staging times and enhanced aeration on the removal of carbon, nitrogen, and phosphorous from cheese production wastewaters. Increasing anaerobic staging times was found to improve the removal rates of carbon beyond previously reported moving bed biofilm reactor results. Increasing the anaerobic stage however decreased the total nitrogen removal, with organic nitrogen undergoing ammonification during the anaerobic stage. This study demonstrates an optimum anaerobic staging time of 138 min; with a carbon removal rate of 31.1 g-sCOD·m -2 d -1 and a nitrogen removal rate of 1.3 g-N·m -2 d -1 . Enhanced aeration was found to be detrimental to phosphorous removal, where a moderate aeration rate demonstrated a net total phosphorous removal of approximately 22 mg-P·l -1 with the phosphorous-content of the suspended solids being approximately 4%. Finally, the sequencing batch moving bed biofilm reactor shows potential for on-site treatment of carbon, nitrogen, and phosphorous from cheese production wastewater., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Co-digestion samples containing thickened waste activated sludge and fat, oil and grease were subjected to three different pretreatment methods, i.e., microwave at 175 °C, hyper-thermophilic stage at 70 °C, and conventional heat at 70 °C. The soluble matter extracted from the un-pretreated and pretreated samples were subjected to an ultrafiltration (UF) process using four different membrane sizes (300, 100, 10, and 1 kDa) for molecular weight distribution analysis. Every pretreatment method had a different effect on the solubilization and redistribution of the soluble matter (SCOD and TVFA). For example while MW pretreatment resulted in a significant increase in the SCOD at the lowest molecular weight (< 1 kDa) and at the highest molecular weight (> 300 kDa), Hyper pretreatment caused the majority of the SCOD ( ̴ 62.7% of total SCOD) to be concentrated at the smaller molecular weight range (< 10 kDa). The MW and hyper-thermophilic pretreatments were much more effective in increasing samples solubilization and biogas production compared to the conventional heat pretreatment. The hyper-thermophilic samples had the maximum improvement in cumulative biogas production from all the molecular weights compared to MW- and Heat-pretreated samples; Hyper-pretreated samples achieved 86.5% higher cumulative biogas production compared to the control.