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Reclaimed water poses environmental and human health risks due to residual organic micropollutants and pathogens. Ozonation of reclaimed water to control pathogens and trace organics is an important step in advanced water treatment systems for potable reuse of reclaimed water. Ensuring efficient pathogen reduction while controlling disinfection byproducts remains a significant challenge to implementing ozonation in reclaimed water reuse applications. This study aimed to investigate ozonation conditions using a plug flow reactor (PFR) to achieve effective pathogen removal/inactivation while minimizing bromate and N-Nitrosodimethylamine (NDMA) formation. The pilot scale study was conducted using three doses of ozone (0.7, 1.0 and 1.4 ozone/total organic carbon (O3/TOC) ratio) to determine the disinfection performance using actual reclaimed water. The disinfection efficiency was assessed by measuring total coliforms, Escherichia coli (E. coli), Pepper Mild Mottle Virus (PMMoV), Tomato Brown Rugose Fruit Virus (ToBRFV) and Norovirus (HNoV). The ozone CT values ranged from 1.60 to 13.62 mg min L-1, resulting in significant reductions in pathogens and indicators. Specifically, ozone treatment led to concentration reductions of 2.46-2.89, 2.03-2.18, 0.46-1.63, 2.23-2.64 and > 4 log for total coliforms, E. coli, PMMoV, ToBRFV, and HNoV, respectively. After ozonation, concentrations of bromate and NDMA increased, reaching levels between 2.8 and 12.0 µg L-1, and 28-40.0 ng L-1, respectively, for average feed water bromide levels of 86.7 ± 1.8 µg L-1 and TOC levels of 7.2 ± 0.1 mg L-1. The increases in DBP formation were pronounced with higher ozone dosages, possibly requiring removal/control in subsequent treatment steps in some potable reuse applications.

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Solar disinfection (SODIS) is an affordable and sustainable Household Water Treatment (HWT) method endorsed by WHO. However, its limitations include longer sunlight exposure requirements, incomplete microbial inactivation, and post-SODIS microbial regrowth during monsoon and winter seasons in subtropical climates. To address these limitations, the performance of SODIS with H2O2 for microbial inactivation during the monsoon and winter seasons in Bangladesh was evaluated following the WHO HWT protocols. Moreover, the process was verified using drinking water samples collected from restaurants, households, and slums. All SODIS experiments were conducted using reflective reactors with PET bottles and plastic bags, adding 10 mg/L of H2O2, and exposing them to sunlight for 6 h. The results showed that E. coli was completely inactivated within 2 h in plastic bags and within 3 h in PET bottles during the monsoon season, achieving an LRV of > 5. In winter, both achieved an LRV > 5 within 3 h and plastic bags showed more efficient in microbial inactivation than PET bottles. The microbial inactivation rates were 5 times higher than those of conventional SODIS. No regrowth of microorganisms was observed during the subsequent post-SODIS period of 12 h and 24 h at room temperature. The study findings suggest that SODIS with H2O2 has the potential for complete microorganism inactivation with shorter sunlight exposure in subtropical climates with moderate to low solar irradiation and can be adopted as a reliable disinfection option for rural and urban communities with unsafe drinking water supply.

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Pathogen reduction for the purpose of human health protection is a critical function provided by water reuse systems. Pathogen reduction performance potential is dependent on a wide range of design and operational parameters. Poor understanding of pathogen reduction performance has important consequences-under treatment can jeopardize human health, while over treatment can lead to unnecessary costs and environmental impacts. Documented pathogen reduction potential of the unit processes that make up water reuse treatment trains is based on a highly dispersed and unstructured literature, creating an impediment to practitioners looking to design, model or simply better understand these systems. This review presents a database of compiled log reduction values (LRVs) and log reduction credits (LRCs) for unit processes capable of providing some level of pathogen reduction, with a focus on processes suitable for onsite non-potable water reuse systems. Where reported, we have also compiled all relevant design and operational factors associated with the LRVs and LRCs. Overall, we compiled over 1100 individual LRV data entries for 31 unit processes, and LRCs for 8 unit processes. Results show very inconsistent reporting of influencing parameters, representing a limitation to the use of some of the data. As a standalone resource, the database (included as Supplemental Information) provides water reuse practitioners with easy access to LRV and LRC data. The database is also part of a longer-term effort to optimize the balance between human health protection, potential environmental impacts and cost of water reuse treatment trains.

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The potential risk of SARS-CoV-2 in treated effluent from a wastewater treatment plant (WWTP) is concerned since SARS-CoV-2 is contained in wastewater during the COVID-19 outbreak. However, the removal of SARS-CoV-2 in WWTP has not been well investigated. The objectives of this study were (i) to clarify the removal performance of SARS-CoV-2 during wastewater treatment, (ii) to compare the removal performance of different secondary treatment processes, and (iii) to evaluate applicability of pepper mild mottle of virus (PMMoV) as a performance indicator for the reduction of SARS-CoV-2 RNA in wastewater treatment. Influent wastewater, secondary-treatment effluent (before chlorination), and final effluent (after chlorination) samples were collected from a WWTP from May 28 to September 24, 2020, during the COVID-19 outbreak in Japan. The target WWTP had three parallel treatment systems employing conventional activated sludge (CAS), anaerobic-anoxic -oxic (A2O), and membrane bioreactor (MBR) processes. SARS-CoV-2 in both the liquid and solid fractions of the influent wastewater was concentrated and quantified using RT-qPCR. SARS-CoV-2 in treated effluent was concentrated from 10 L samples to achieve a detection limit as low as 10 copies/L. The log reduction value (LRV) of SARS-CoV-2 was 2.7 ± 0.86 log10 in CAS, 1.6 ± 0.50 log10 in A2O, and 3.6 ± 0.62 log10 in MBR. The lowest LRV observed during the sampling period was 2.8 log10 in MBR, 1.2 log10 in CAS, and 1.0 log10 in A2O process, indicating that the MBR had the most stable reduction performance. PMMoV was found to be a good indicator virus to evaluate reduction performance of SARS-CoV-2 independent of the process configuration because the LRV of PMMoV was significantly lower than that of SARS-CoV-2 in the CAS, A2O and MBR processes.

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Treatment of wastewater for potable reuse is increasingly becoming a suitable alternative water source to meet the growing urban water needs worldwide. Potable reuse requires reduction of enteric viruses to levels at which they do not pose a risk to human health. Advanced water treatment trains (e.g., microfiltration (MF), ultrafiltration (UF), reverse osmosis (RO), and ultraviolet light and advanced oxidation process (UV/AOP)) provide significant protection and reduce virus loads in highly treated final product waters. Even though viruses are a principal concern, the performance of virus removal by membrane processes is not easily determined. The objective of this study was to evaluate the applicability of Aichi virus (AiV), pepper mild mottle virus (PMMoV), cucumber green mottle mosaic virus (CGMMV), and cross-assembly phage (crAssphage) removal as possible process indicators for MF, UF, and RO. Virus log reduction values (LRVs) based on gene copies measured using molecular methods were determined for MF and UF. The median LRVs of all viruses obtained after MF and UF were 2.9 and 3.1, respectively. The LRVs of the proposed indicators were lower than those of human enteric viruses. The morphological and physicochemical difference among indicators was not found to affect LRVs. Therefore, all proposed indicator viruses were determined to be suitable candidates as process indicators for MF and UF. Regarding RO, most of the viruses measured in this study were undetectable in permeate. Only PMMoV and CGMMV were detected showing median LRVs of 2.8 and 2.5, respectively. PMMoV and CGMMV are recommended as good process indicators of physical virus removal for the overall water treatment process.

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BACKGROUND AIMS: Platelet concentrates (PCs) are pooled to prepare human platelet lysate (HPL) supplements of growth media to expand primary human cells for transplantation; this increases the risk of contamination by known, emerging, and unknown viruses. This possibility should be of concern because viral contamination of cell cultures is difficult to detect and may have detrimental consequences for recipients of cell therapies. Viral reduction treatments of chemically defined growth media have been proposed, but they are not applicable when media contain protein supplements currently needed to expand primary cell cultures. Recently, we successfully developed a Planova 35NPlanova 20N nanofiltration sequence of growth media supplemented with two types of HPL. The nanofiltered medium was found to be suitable for mesenchymal Stromal cell (MSC) expansion. METHODS: Herein, we report viral clearance achieved by this nanofiltration process used for assessing a new experimental model using non-infectious minute virus of mice-mock virus particle (MVM-MVP) and its quantification by an immunoqPCR. Then, high doses of MVM-MVP (1012 MVPs/mL) were spiked to obtain a final concentration of 1010 MVPs/mL in Planova 35N-nanofiltered growth medium supplemented with both types of HPLs [serum converted platelet lysate SCPL) and intercept human platelet lysate (I-HPL)] at 10% (v/v) and then filtering through Planova 20N. RESULTS: No substantial interference of growth medium matrices by the immune-qPCR assay was first verified. Log reduction values (LRVs) were ≥ 5.43 and ≥ 5.36 respectively, SCPL and I-HPL media. MVM-MVPs were also undetectable by dynamic light scattering and transmission electron microscopy. CONCLUSIONS: The nanofiltration of growth media supplemented with 10% HPL provides robust removal of small nonenveloped viruses, and is an option to improve the safety of therapeutic cells expanded using HPL supplements.

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Wastewater reclamation and reuse have been practically applied to water-stressed regions, but waterborne pathogens remaining in insufficiently treated wastewater are of concern. Sanitation Safety Planning adopts the hazard analysis and critical control point (HACCP) approach to manage human health risks upon exposure to reclaimed wastewater. HACCP requires a predetermined reference value (critical limit: CL) at critical control points (CCPs), in which specific parameters are monitored and recorded in real time. A disinfection reactor of a wastewater treatment plant (WWTP) is regarded as a CCP, and one of the CCP parameters is the disinfection intensity (e.g., initial disinfectant concentration and contact time), which is proportional to the log reduction value (LRV) of waterborne pathogens. However, the achievable LRVs are not always stable because the disinfection intensity is affected by water quality parameters, which vary among WWTPs. In this study, we established models for projecting virus LRVs using ozone, in which water quality and operational parameters were used as explanatory variables. For the model construction, we used five machine learning algorithms and found that automatic relevance determination with interaction terms resulted in better prediction performances for norovirus and rotavirus LRVs. Poliovirus and coxsackievirus LRVs were predicted well by a Bayesian ridge with interaction terms and lasso with quadratic terms, respectively. The established models were relatively robust to predict LRV using new datasets that were out of the range of the training data used here, but it is important to collect LRV datasets further to make the models more predictable and flexible for newly obtained datasets. The modeling framework proposed here can help WWTP operators and risk assessors determine the appropriate CL to protect human health in wastewater reclamation and reuse.

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The world is combating the emergence of Coronavirus disease 2019 (COVID-19) caused by novel coronavirus; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Further, due to the presence of SARS-CoV-2 in sewage and stool samples, its transmission through water routes cannot be neglected. Thus, the efficient treatment of wastewater is a matter of utmost importance. The conventional wastewater treatment processes demonstrate a wide variability in absolute removal of viruses from wastewater, thereby posing a severe threat to human health and environment. The fate of SARS-CoV-2 in the wastewater treatment plants and its removal during various treatment stages remains unexplored and demands immediate attention; particularly, where treated effluent is utilised as reclaimed water. Consequently, understanding the prevalence of pathogenic viruses in untreated/treated waters and their removal techniques has become the topical issue of the scientific community. The key objective of the present study is to provide an insight into the distribution of viruses in wastewater, as well as the prevalence of SARS-CoV-2, and its possible transmission by the faecal-oral route. The review also gives a detailed account of the major waterborne and non-waterborne viruses, and environmental factors governing the survival of viruses. Furthermore, a comprehensive description of the potential methods (physical, chemical, and biological) for removal of viruses from wastewater has been presented. The present study also intends to analyse the research trends in microalgae-mediated virus removal and, inactivation. The review also addresses the UN SDG 'Clean Water and Sanitation' as it is aimed at providing pathogenically safe water for recycling purposes.

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A narrow residence time distribution (RTD) is highly desirable for continuous processes where a strict incubation time must be ensured, such as continuous virus inactivation. A narrow RTD also results in faster startup and shut down phases and limits the broadening of potential disturbances in continuous processes. A packed bed reactor with non-porous inert beads was developed to achieve narrow RTDs. The performance was defined as the ratio between the onset of the cumulative RTD and the median residence time (tx%/t50%). Laboratory-scale packed columns were used to study the influence of the column parameters on the RTD. A larger column with a void volume of 0.65 L and a length of 89 cm, packed with beads in a size range of 125 to 250 µm, achieved t0.5%/t50% >0.93 across flow rates from 0.1 to 9.8 mL/min. The RTD was significantly narrower than the RTDs of other reactor designs, such as the Coiled Flow Inverter and Jig in a Box. The pressure drop remained under 3 kPa for all tested flow rates. Fluorescent nanoparticles (30 and 200 nm) were used to mimic viruses. These two sizes showed less than 2% difference in terms of t1%/t50% and t0.01%/t50% scores. These results indicated that viruses travelled through the column at rates independent of size. This proposal of packed beds as incubation chambers for continuous virus inactivation is simple, scalable, and can be realized as single-use devices. Due to the low pressure drop, the system can be easily integrated into a fully continuous process.

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Low pH inactivation of enveloped viruses has historically been shown to be an effective viral inactivation step in biopharmaceutical manufacturing. To date, most statistical analyses supporting modular low pH viral inactivation claims have used descriptive statistical analyses, which in many cases do not allow for probabilistic characterization of future experimental log10 reduction values (LRVs). Using Bayesian hierarchical logistic regression modeling, probability statements regarding the likelihood of successful low pH viral inactivation based on only certain process parameter settings can be derived. This type of analysis also permits statistical modeling in the presence of historical data from different experiments and right-censored data, two issues that have not as yet been satisfactorily dealt with in the literature. The characterization of the probability of successful inactivation allows creation of a modular claim stating future LRVs will be greater than or equal to some critical value, based on only certain process parameter settings of the viral inactivation unit operation. This risk-based approach, when used in conjunction with traditional descriptive statistics, facilitates coherent and cogent decision-making about modular viral clearance LRV claims.LAY ABSTRACT: Viral contamination of biologically derived drug products is a safety concern for both regulatory agencies and drug manufacturers. Validation of the removal and inactivation of model viruses is required to ensure the safety of patients receiving these drugs, and dedicated steps, including viral filtration and chemical inactivation, are often added to manufacturing processes to provide additional clearance and inactivation capabilities. One of these steps, low pH inactivation, exposes enveloped viruses to a low pH environment to reduce the potential of the virus to infect host cells. Because the viral inactivation capability of this well-understood unit operation has been demonstrated for years across many different biological drugs, many companies have begun investigating the use of the modular viral clearance claim for the low pH inactivation step. Modular claims ensure, without experimentation, that a certain level of reduction of virus will occur if specific parameters are used in the manufacturing process, allowing manufacturers to save both time and resources in the early developmental phases of biologically derived drugs. A novel type of statistical analysis is outlined in this article that when used in addition to previously used analyses allows drug manufacturers to estimate a more valid level of virus reduction in modular viral clearance claims.

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The reuse of water in a range of potable and non-potable applications is an important factor in the augmentation of water supply and in improving water security and productivity worldwide. A key hindrance to the reuse of water is the cost of compliance testing and process validation associated with ensuring that pathogen and chemicals in the feedwater are removed to a level that ensures no acute or chronic health and/or environmental effects. The critical control point (CCP) approach is well established and widely adopted by water utilities to provide an operational and risk management framework for the removal of pathogens in the treatment system. The application of a CCP approach to barriers in a treatment system for the removal of chemicals is presented. The application exemplar is to a small community wastewater treatment system that aims to produce potable quality water from a secondary treated wastewater effluent, however, the concepts presented are generic. The example used seven treatment barriers, five of which were designed and operated as CCP barriers for pathogens. The work demonstrates a method and risk management framework by which three of the seven barriers could also include a CCP approach for the removal of chemicals. Analogous to a CCP approach for pathogens, the potential is to reduce the use of chemical analysis as a routine determinant of performance criteria. The operational deployment of a CCP approach for chemicals was augmented with the development of a decision tree encompassing the classification of chemicals and the total removal credits across the treatment train in terms of the mechanistic removal of chemicals for each barrier. Validation of the approach is shown for an activated sludge, ozone and reverse osmosis barrier.

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The LRVs required to decrease HE concentrations in raw sewage to an acceptable level to manage the risk to human and livestock health were determined. An LRV of 3.0 was required to meet the HBT of 1 µDALY pppy in SE Australia where human helminth infections are not endemic. In comparison, a similar exposure volume and LRV in endemic regions would result in a HBT of 100 µDALY pppy. The risks posed by cattle- and pig-related helminths were also managed acceptably with the treatment of sewage providing an LRV of 3.0. New design equations were derived to determine LRVs based on hydraulic residence times (HRTs) in an activated sludge plant (ASP) and lagoons. The new equation for lagoons indicated that an LRV of 3.0 could be achieved with a HRT of 18 days or less.

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Ultrafiltration is an effective barrier to waterborne pathogens including viruses. Challenge testing is commonly used to test the inherent reliability of such systems. Performance validation seeks to demonstrate the adequate reliability of the treatment system. Appropriate and rigorous data analysis is an essential aspect of validation testing. In this study we used Bayesian analysis to assess the performance of a full-scale ultrafiltration system which was validated and revalidated after five years of operation. A hierarchical Bayesian model was used to analyse a number of similar ultrafiltration membrane skids working in parallel during the two validation periods. This approach enhanced our ability to obtain accurate estimations of performance variability, especially when the sample size of some system skids was limited. This methodology enabled the quantitative estimation of uncertainty in the performance parameters and generation of predictive distributions incorporating those uncertainties. The results indicated that there was a decrease in the mean skid performance after five years of operation of approximately 1 log reduction value (LRV). Interestingly, variability in the LRV also reduced, with standard deviations from the revalidation data being decreased by a mean 0.37 LRV compared with the original validation data. The model was also useful in comparing the operating performance of the various parallel skids within the same year. Evidence of differences was obtained in 2015 for one of the membrane skids. A hierarchical Bayesian analysis of validation data provides robust estimations of performance and the incorporation of probabilistic analysis which is increasingly important for comprehensive quantitative risk assessment purposes.

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The purification of antibodies by precipitating impurities using Polyethylene Glycol (PEG) was assessed with the objective of developing a two chromatography column purification process. A PEG precipitation method was evaluated for use in the industrial purification of recombinant monoclonal antibodies (MAbs). Effective and robust precipitation conditions including PEG concentration, pH, temperature, time, and protein concentration were identified for several different MAbs. A recovery process using two chromatography steps in combination with PEG precipitation gave acceptable yield and purity levels for IgG1 and IgG4 antibodies with a broad range of isoelectric points (pI). PEG precipitation removed host cell proteins (HCPs), high molecular weight species (HMWS), leached Protein A ligand, and host cell DNA to acceptable levels when run under appropriate conditions, and some endogenous virus removal was achieved.

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