Search

Wastewater-based surveillance (WBS) is increasingly used for monitoring disease targets in wastewaters around the world. This study, performed in Ottawa, Canada, identifies a decrease in SARS-CoV-2 wastewater measurements during snowmelt-induced sewer flushing events. Observations first revealed a correlation between suppressed viral measurements and periods of increased sewage flowrates, air temperatures above 0 °C during winter months, and solids mass flux increases. These correlations suggest that high sewage flowrates from snowmelt events or intense precipitation events lead to the scouring of previously settled solids in sewers and the subsequent entrainment of these solids into the transported wastewaters. Collection of WBS samples during flushing events hence contains a heterogeneous mixture of solids, including resuspended solids with varying degrees of decay. Therefore flushing events can present a challenge for accurately measuring disease target viral signals when using solids-based analytical methods. This study demonstrates that resuspended solids entrained in the wastewaters during flushing events retain PMMoV signal while the SARS-CoV-2 signal is significantly reduced due to the slower decay rate of pepper mild mottle virus (PMMoV) compared to SARS-CoV-2 within wastewaters. Hence current normalization methods using PMMoV are shown to be ineffective in correcting for flushing events and the associated resuspension of settled solids, as the PMMoV signal of settled solids within sewers does not account for the differential decay rates experiences by SARS-CoV-2 signal in settled solids. Instead, this study identifies RNA to PMMoV correction factor as an effective approach to correct for flushing events and to realign SARS-CoV-2 signal with COVID-19 hospital admission rates within communities. As such, the study highlights the key physicochemical parameters necessary to identify flushing events that affect SARS-CoV-2 WBS measurements and introduces a novel RNA to PMMoV correction factor approach for solids-based analysis of SARS-CoV-2 during flushing events, enhancing the accuracy of WBS data for public health decision-making., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

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 The Authors. Published by Elsevier GmbH.. 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.

The effects of microwave (MW) and combined alkaline-MW pretreatments on the co-digestion of TWAS:FOG mixtures with 20, 40 and 60% FOG were investigated. MW pretreatment at a high temperature of 175ᵒC was shown to be the most effective MW pretreatment option in solubilizing TWAS:FOG mixtures and boosting methane yield. MW pretreatment at 175ᵒC resulted in maximum solubilization (%) of 68.2% for the 20%FOG samples and a maximum methane yield that was 137% higher than the control for samples with 60%FOG. The combined alkaline-MW (NaOH-MW) pretreatment at pH 10 proved to be not an effective option for TWAS:FOG pretreatment before the anaerobic co-digestion. Despite the benefits of MW pretreatment on the TWAS:FOG samples, including a significant increase in solubilization, dewaterability improvement, high VS reductions, and high methane yield productions, the energy analysis resulted in negative net energy values for all MW-pretreated samples., (Copyright © 2018. Published by Elsevier Ltd.)

Two sequencing batch reactors (SBRs) were operated for 100days under aerobic conditions, with one being fed with unsterilized municipal wastewater (USBR), and the other fed with sterilized municipal wastewater (SSBR). Respirometric assays and fluorescence in situ hybridization (FISH) results show that active nitrifiers were present in the unsterilized influent municipal wastewater. The maximum ammonia utilization rate (AUR) and nitrite utilization rate (NUR) of the unsterilized influent were 0.32±0.12mg NH 4 + -N/(L·hr) and 0.71±0.18mg NO 2 - -N/(L·hr). Based on the maximum utilization rates, the estimated seeding intensity for the ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) of the USBR was 0.08g AOB/(g AOB·day) and 0.20g NOB/(g NOB·day) respectively. The fraction of nitrifiers/total bacteria in the influent was 5.35%±2.1%, the dominant AOB was Nitrosomonas spp., Nitrosococcus mobilis hybridizated with Nsm156, and the dominant NOB was Nitrospira hybridizated with Ntspa662. The influent nitrifiers potentially seeded the activated sludge of the bioreactor and hence demonstrated a mitigation of the acclimatization times and instability during start-up and early operation. The AUR and NUR in the USBR was 15% and 13% higher than the SSBR respectively during the stable stage, FISH results showed that nitrifiers population especially the Nitrospira in the USBR was higher than that in the SSBR. These results indicate that the natural continuous immigration of nitrifiers from municipal influent streams may have some repercussions on the modeling and design of bioreactors., (Copyright © 2017. Published by Elsevier B.V.)

Renewable energy and clean environment are two crucial requirements for our modern world. Low cost, energy production and limited environmental impact make anaerobic digestion (AD) a promising technology for stabilizing organic waste and in particular, sewage waste. The anaerobic co-digestion of thickened waste activated sludge (TWAS) and sewage treatment plant trapped fat, oil and grease (FOG) using different FOG-TWAS mixtures (20, 40, 60 and 80% of FOG based on total volatile solids (TVS)) were investigated in this study using both thermophilic (55 ± 1 °C) and two stages hyper-thermophilic/thermophilic (70 ± 1 °C and 55 ± 1 °C) anaerobic co-digestion. The hyper-thermophilic co-digestion approach as a part of the co-digestion process has been shown to be very useful in improving the methane production. During hyper-thermophilic biochemical methane potential (BMP) assay testing the sample with 60% FOG (based on TVS) has been shown to significantly increase the maximum methane production to 673.1 ± 14.0 ml of methane as compared to 316.4 ± 14.3 ml of methane for the control sample. This represents a 112.7% increase in methane production compared to the control sample considered in this paper. These results signify the importance of hyper-thermophilic digestion to the co-digestion of TWAS-FOG field., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The objective of this study is to investigate and optimize the co-digestion of scum with thickened waste activated sludge (TWAS) and primary sludge (PS) undergoing mesophilic anaerobic digestion. The effect of scum loading on the co-digestion of PS, TWAS and scum has shown to have a significant impact on the ultimate cumulative biogas production and on the specific biogas production between 20 and 40 days of digestion, while the effects of the scum holding time within the scum concentrator and temperature of the scum concentrator did not demonstrate a significant effect on the ultimate or specific biogas production. The study demonstrates that care must be taken to avoid inhibitory effects and potential souring of digesters due to scum overloading and specifically scum overloading in combination with long holding times of scum within the scum concentrator at elevated temperatures.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Fe3+ 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 Fe3+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The moving bed biofilm reactor is a compact technology established for treating total ammonia nitrogen (TAN) from municipal wastewater via nitrification or denitrification. The sequencing batch moving bed biofilm reactor (SB-MBBR) has been applied for on-site biological treatment of carbon and phosphorous from cheese production wastewater; however, nitrification is limited by the competition between nitrifiers and heterotrophs. Two strategies are compared to circumvent heterotrophic competition and achieve TAN oxidation in an SB-MBBR system already achieving carbon and phosphorous removal: extended aerobic operation of a single SB-MBBR and two SB-MBBRs in series. TAN oxidation occurred after 810 hours with the extended aerobic operation, where a microbiome shift occurred to support an ammonia-oxidizing bacterial population. Thus, a single SB-MBBR is not feasible for achieving nitrification simultaneously with carbon and phosphorous removal when treating cheese production wastewater. After 30 hours of operation, two SB-MBBRs in series achieve TAN removal, possibly through partial nitritation, with a TAN surface area removal rate of 1.07 ± 0.05 g-N·m−2 d −1 and an enriched abundance of ammonia-oxidizing bacteria. To the best of our knowledge, this is the first study to analyze the microbiome of the SB-MBBR achieving TAN removal from cheese production wastewater and present an operational strategy to achieve TAN while treating cheese production wastewater with SB-MBBRs. Also, this is the first study to show evidence that partial nitrification can be achieved in an SB-MBBR system that is also treating carbon and phosphorous from cheese production wastewater and demonstrates the potential for the SB-MBBR to be incorporated in a deammonification system. 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. [ABSTRACT FROM AUTHOR]

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. [Display omitted] [ABSTRACT FROM AUTHOR]

• Incidence proportion of Variants of Concern can be estimated via wastewater. • Allele and lineage specificity is possible with single locus primer extension qRT-PCR. • High correlation of VOC-positive clinical cases and VOC-specific wastewater signal. • Allows near real-time monitoring of VOC infection dynamics in a population. 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. [Display omitted] [ABSTRACT FROM AUTHOR]