Omicron COVID-19 Case Estimates Based on Previous SARS-CoV-2 Wastewater Load, Regional Municipality of Peel, Ontario, Canada.

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.

The microbiome of two strategies for ammonia removal with the sequencing batch moving bed biofilm reactor treating cheese production wastewater.

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.

Rapidly developed, optimized, and applied wastewater surveillance system for real-time monitoring of low-incidence, high-impact MPOX outbreak.

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.

Influence of MBBR carrier geometrical properties and biofilm thickness restraint on biofilm properties, effluent particle size distribution, settling velocity distribution, and settling behaviour.

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/(m2 •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/m3). The K5 effluent solids also showed enhanced settling behaviour, consisting of larger particles with faster settling velocities.

Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada.

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-log10 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-log10 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.

Partial nitritation at elevated loading rates: design curves and biofilm characteristics.

There is a need to develop low operational intensity, cost-effective, and small-footprint systems to treat wastewater. Partial nitritation has been studied using a variety of control strategies, however, a gap in passive operation is evident. This research investigates the use of elevated loading rates as a strategy for achieving low operational intensity partial nitritation in a moving bed biofilm reactor (MBBR) system. The effects of loading rates on nitrification kinetics and biofilm characteristics were determined at elevated, steady dissolved oxygen concentrations between 5.5 and 7.0 mg O2/L and ambient temperatures between 19 and 21 °C. Four elevated loading rates (3, 4, 5 and 6.5 g NH4+-N/m2 days) were tested with a distinct shift in kinetics being observed towards nitritation at elevated loadings. Complete partial nitritation (100% nitrite production) was achieved at 6.5 g NH4+-N/m2 days, likely due to thick biofilm (572 µm) and elevated NH4+-N load, which resulted in suppression of nitrite oxidation.

Microwave vs. alkaline-microwave pretreatment for enhancing Thickened Waste Activated Sludge and fat, oil, and grease solubilization, degradation and biogas production.

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.

Predicting wastewater treatment plant quality parameters using a novel hybrid linear-nonlinear methodology.

Biochemical oxygen demand (BOD), chemical oxygen demand (COD), total dissolved solids (TDS) and total suspended solids (TSS) are the most commonly regulated wastewater effluent parameters. The measurement and prediction of these parameters are essential for assessing the performance and upgrade of wastewater treatment facilities. In this study, a new methodology, combining a linear stochastic model (ARIMA) and nonlinear outlier robust extreme learning machine technique (ORELM) with various preprocesses, is presented to model the quality parameters of effluent wastewater (ARIMA-ORELM). For each of the studied parameters, 144 different (144 × 8 models) linear models (ARIMA) are presented, with the superior model of each parameter being selected based on statistical indices. Moreover, 48 nonlinear models (ORELM) and 48 hybrid models (ARIMA-ORELM) were considered. The use of linear and nonlinear approaches to model the linear and nonlinear terms (respectively) of each time series in the hybrid model increased the efficiency and accuracy of the predictions for all of the time series. The influent wastewater nonlinear TSS model and the effluent COD and BOD models attained the best performance with a high correlation coefficient of 0.95. The use of hybrid models improved the prediction capability of all quality parameters with the best performance being achieved for the effluent BOD model (R2 = 0.99).

Investigation of copper inhibition of nitrifying moving bed biofilm (MBBR) reactors during long term operations.

Copper, a prevalent heavy metal in industrial mining wastewaters, has been shown to inhibit nitrification in wastewater treatment systems. Biofilm treatment systems have an inherent potential to reduce inhibition. This study investigated the effects of copper concentration on nitrifying biofilms in moving bed biofilm reactor (MBBR) systems across long term operation using influent ammonia concentrations representative of gold mining wastewater. Conventional isotherm models did not adequately model the attachment of copper to the biofilm. Long term nitritation was shown to be uninhibited at influent copper concentrations between 0.13 and 0.61 mg Cu/L. Nitratation was inhibited with influent copper concentrations of 0.28-0.61 mg Cu/L. There was no statistical difference in biofilm characteristics, including biofilm thickness, mass and density, across all copper concentrations tested, however, changes in biofilm morphology were observed. The demonstrated resistance of the nitrifying biofilm to copper inhibition makes the MBBR system a promising technology for treating ammonia in mining wastewaters.

Improving biogas production from anaerobic co-digestion of Thickened Waste Activated Sludge (TWAS) and fat, oil and grease (FOG) using a dual-stage hyper-thermophilic/thermophilic semi-continuous reactor.

This paper investigates the feasibility and advantages of using a dual-stage hyper-thermophilic/thermophilic semi-continuous reactor system for the co-digestion of Thickened Waste Activated Sludge (TWAS) and Fat, Oil and Grease (FOG) to produce biogas in high quantity and quality. The performance of the dual-stage hyper-thermophilic (70°C)/thermophilic (55°C) anaerobic co-digestion system is evaluated and compared to the performance of a single-stage thermophilic (55°C) reactor that was used to co-digest the same FOG-TWAS mixtures. Both co-digestion reactors were compared to a control reactor (the control reactor was a single-stage thermophilic reactor that only digested TWAS). The effect of FOG% in the co-digestion mixture (based on total volatile solids) and the reactor hydraulic retention time (HRT) on the biogas/methane production and the reactors' performance were thoroughly investigated. The FOG% that led to the maximum methane yield with a stable reactor performance was determined for both reactors. The maximum FOG% obtained for the single-stage thermophilic reactor at 15 days HRT was found to be 65%. This 65% FOG resulted in 88.3% higher methane yield compared to the control reactor. However, the dual-stage hyper-thermophilic/thermophilic co-digestion reactor proved to be more efficient than the single-stage thermophilic co-digestion reactor, as it was able to digest up to 70% FOG with a stable reactor performance. The 70% FOG in the co-digestion mixture resulted in 148.2% higher methane yield compared to the control at 15 days HRT. 70% FOG (based on total volatile solids) is so far the highest FOG% that has been proved to be useful and safe for semi-continuous reactor application in the open literature. Finally, the dual-stage hyper-thermophilic/thermophilic co-digestion reactor also proved to be efficient and stable in co-digesting 40% FOG mixtures at lower HRTs (i.e., 9 and 12 days) and still produce high methane yields and Class A effluents.

Natural continuous influent nitrifier immigration effects on nitrification and the microbial community of activated sludge systems.

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 NH4+-N/(L·hr) and 0.71±0.18mg NO2--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.

Rapid start-up of nitrifying MBBRs at low temperatures: nitrification, biofilm response and microbiome analysis.

The moving bed biofilm reactor (MBBR), operated as a post carbon removal system, requires long start-up times in comparison to carbon removal systems due to slow growing autotrophic organisms. This study investigates the use of carriers seeded in a carbon rich treatment system prior to inoculation in a nitrifying MBBR system to promote the rapid development of nitrifying biofilm in an MBBR system at temperatures between 6 and 8 °C. Results show that nitrification was initiated by the carbon removal carriers after 22 h of operation. High throughput 16S-rDNA sequencing indicates that the sloughing period was a result of heterotrophic organism detachment and the recovery and stabilization period included a growth of Nitrosomonas and Nitrospira as the dominant ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in the biofilm. Peripheral microorganisms such as Myxococcales, a rapid EPS producer, appear to have contributed to the recovery and stabilization of the biofilm.

Thermophilic and hyper-thermophilic co-digestion of waste activated sludge and fat, oil and grease: Evaluating and modeling methane production.

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.

Investigation of settleability of biologically produced solids and biofilm morphology in moving bed bioreactors (MBBRs).

The objective of this work is to investigate the effects of surface area loading rates (SALRs) and hydraulic retention times (HRTs) in moving bed bioreactor (MBBR) systems on the morphology and thickness of the attached biofilm along with subsequent effects on particle size distribution and the settling characteristics of the biologically produced solids. The morphology of biofilm attached to the MBBR carriers changed from a porous biofilm to a biofilm with a more filamentous structure throughout the study at various operating conditions without observable correlation with SALR and HRT. Although, biofilm morphology did not demonstrate an effect on the biologically produced solids observed in this study, the thinnest biofilms resulted in the highest concentration of solids in the effluent. Furthermore, the particle size distribution analysis demonstrated that both higher SALRs and longer HRTs resulted in a shift towards larger-sized particles. Increases in SALR and HRT, independent of each other, also showed increases in effluent solid concentration and lower settleability of the solids.

An investigation of moving bed biofilm reactor nitrification during long-term exposure to cold temperatures.

Biological treatment is the most common and economical means of ammonia removal in wastewater; however, nitrification rates can become completely impeded at cold temperatures. Attached growth processes and, specifically, moving bed biofilm reactors (MBBRs) have shown promise with respect to low-temperature nitrification. In this study, two laboratory MBBRs were used to investigate MBBR nitrification rates at 20, 5, and 1 degree C. Furthermore, the solids detached by the MBBR reactors were investigated and Arrhenius temperature correction models used to predict nitrification rates after long-term low-temperature exposure was evaluated. The nitrification rate at 5 degrees C was 66 +/- 3.9% and 64 +/- 3.7% compared to the rate measured at 20 degrees C for reactors 1 and 2, respectively. The nitrification rates at 1 degree C over a 4-month exposure period compared to the rate at 20 degrees C were 18.7 +/- 5.5% and 15.7 +/- 4.7% for the two reactors. The quantity of solids detached from the MBBR biocarriers was low and the mass of biofilm per carrier did not vary significantly at 20 degrees C compared to that after long-term exposure at 1 degree C. Lastly, a temperature correction model based on exposure time to cold temperatures showed a strong correlation to the calculated ammonia removal rates relative to 20 degrees C following a gradual acclimatization period to cold temperatures.

Field study of moving bed biofilm reactor technology for post-treatment of wastewater lagoon effluent at 1 degree C.

The goal of this study was to investigate the potential use ofmoving bed biofilm reactor (MBBR) systems as ammonia removal post-treatment units for wastewater (WW) treatment lagoons that demonstrate large temperature changes throughout their operational year (1 - 20 degrees C). The study was carried out over a six-month period using laboratory-scale MBBR reactors fed with incoming effluent from a full-scale lagoon. The study shows that significant average ammonia removal rates of 0.26 and 0.11 kgN/m . d were achieved at 20 degrees C and 1C. The increase in the ammonia removal rates with increasing temperature from 1 degrees C to 20 degrees C showed a strong correlation to an applied temperature correction coefficient model. No significant accumulation of effluent nitrite was observed at 1 degrees C or after being fed with synthetic wastewater (SWW); indicating that cold temperatures and transitions from real WW to SWW did not stress the nitrifiers. Furthermore, the study demonstrates that changes in temperature or changes from real WW to SWW do not affect the mass of biofilm attached per MBBR carrier. Hence, based on the results of this study, it is concluded that MBBR is a promising technology for post-treatment ammonia removal of WW lagoon effluent.

Real-time evaluation of signal accuracy in wastewater surveillance of pathogens with high rates of mutation.

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.

Impact of coagulation on SARS-CoV-2 and PMMoV viral signal in wastewater solids.

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.

Corrigendum: Wastewater-based surveillance identifies start to the pediatric respiratory syncytial virus season in two cities in Ontario, Canada.

[This corrects the article DOI: 10.3389/fpubh.2023.1261165.].

SARS-CoV-2 viral titer measurements in Ontario, Canada wastewaters throughout the COVID-19 pandemic.

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.