Observed Kinetics of Enterovirus Inactivation by Free Chlorine Are Host Cell-Dependent.

Virucidal efficacies of disinfectants are typically assessed by infectivity assay utilizing a single type of host cell. Enteroviruses infect multiple host cells via various entry routes, and each entry route may be impaired differently by a given disinfectant. Yet, it is unknown how the choice of host cells affects the observed inactivation kinetics. Here, we evaluated the inactivation kinetics of echovirus 11 (E11) by free chlorine, ultraviolet (UV) irradiation, and heat, using three different host cells (BGMK, RD, and A549). Inactivation rates were independent of the host cell for treatment of E11 by UV or heat. Conversely, E11 inactivation by free chlorine occurred 2-fold faster when enumerated on BGMK cells compared with RD and A549 cells. Host cell-dependent inactivation kinetics by free chlorine were also observed for echovirus 7, 9, and 13, and coxsackievirus A9. E11 inactivation by free chlorine was partly caused by a loss in host cell attachment, which was most pronounced for BGMK cells. BGMK cells lack the attachment receptor CD55 and a key subunit of the uncoating receptor ß2M, which may contribute to the differential inactivation kinetics for this cell type. Consequently, inactivation kinetics of enteroviruses should be assessed using host cells with different receptor profiles.

Selective elimination of enterovirus genotypes by activated sludge and chlorination.

Enteroviruses, which are commonly circulating viruses shed in the stool, are released into the sewage system and only partially removed or inactivated, resulting in the discharge of infectious enteroviruses into the environment. Activated sludge and chlorination remove or inactivate enterovirus genotypes to different extents, and thus have the potential to shape the population that will be discharged. The goal of this study was to evaluate how activated sludge and chlorination treatment shape an enterovirus population at the genotype level, using a population of eight genotypes commonly found in sewage: CVA9, CVB1, CVB2, CVB3, CVB4, CVB5, E25, E30. Our results show that the extent of inactivation varied among genotypes, but also across sludge samples. We find that the effluent of activated sludge systems will be depleted in CVA9, CVB1 and CVB2 while E25 together with CVB3, CVB4 and CVB5 will be prevalent. Furthermore, we found that microbial inactivation was the main mechanism of infectivity loss in the activated sludge, while adsorption to the sludge flocs was not significant. During effluent chlorination, we also observed that CVB5, CVB3 and to a lesser extent E25 were less susceptible to chlorination while E30 was readily inactivated, and activated sludge-derived EPS provided further protection against chlorination. This study contributes to a better understanding of the variability of sewage treatment efficacy against different enteroviruses.

An integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously quantify the infectious concentrations of eight environmentally relevant enterovirus serotypes.

Enterovirus (EV) infectivity is typically measured as a bulk parameter, yet EV serotypes vary in their susceptibility to natural and engineered stressors. Here we developed an integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method to simultaneously and specifically quantify the infectious concentrations of eight EV serotypes commonly encountered in sewage (coxsackieviruses A9, B1, B2, B3, B4 and B5, and echoviruses 25 and 30). The method uses two cell lines for virus replication and serotype-specific qPCR primers for quantification. Primers were designed to target multiple environmental strains of a given serotype and displayed high specificity. The ICC-RTqPCR method exhibited a linear calibration range between 50 and 1000 (echoviruses) or 5000 (coxsackieviruses) infectious units per mL. Over this range, measurements were not influenced by the presence of non-target serotypes, and calibration slopes were reproducible for different virus batches and cell ages. The ICC-RTqPCR method was able to accurately quantify the infectious concentration of a virus after inactivation by heat, and the concentration of a virus within a wastewater matrix. This method will be valuable to assess the differing fates of EV serotypes in natural or engineered systems, and to portray the associated changes in EV population composition.

Linking transformations of organic carbon to post-treatment performance in a biological water recycling system.

Ozone, electrolysis and granular activated carbon (GAC) were examined as potential post-treatments to follow a household-scale biologically activated membrane bioreactor (BAMBi), treating a wash water containing trace urine and feces contamination. Each post-treatment was evaluated for abilities and reaction preferences to remove or transform dissolved organic carbon (DOC), chemical structures that contribute color, and assimilable organic carbon (AOC), which can support bacterial regrowth. Batch treatment with each technology demonstrated an ability to remove ≥95% DOC. Ozone demonstrated a reaction selectivity through increased reaction rates with larger compounds and color-contributing compounds. Electrolysis and GAC demonstrated generally less-selective reactivity. Adding post-treatments to full-scale systems reduced DOC (55-91%), AOC (34-62%), and color (75-98%), without significant reaction selectivity. These reductions in DOC and AOC were not linked to reduction of bacterial concentrations in treated water. Reductions in bacterial concentrations were observed with ozone and electrolysis, but this is credited to oxidation chemicals produced in these systems and not the removal or transformations of organic materials.

Comparing the anti-bacterial performance of chlorination and electrolysis post-treatments in a hand washing water recycling system.

Innovative solutions are necessary to enable the decentralized recycling of greywater for applications requiring high-quality water, such as hand washing. While physical barriers such as ultrafiltration membranes effectively prevent the passage of bacteria, and chemical and biological treatments can effectively reduce the carbon content of the treated water, there exists a knowledge gap regarding the application of anti-bacterial strategies to prevent the growth of harmful bacteria following treatment. In this study, the effluent water from a household-scale greywater treatment system was fed to seven parallel experimental post-treatment tanks: three receiving direct chlorination with free chlorine residuals of 0.2, 1 or 5 mg Cl2/L, three with chlorine produced through electrolysis at the same residual concentrations, and one control with no chlorine added. For increasing concentrations of direct chlorination, the median total cell count (TCC) values were 9 × 104, 2.9 × 104 and 1.8 × 103 cells/mL, respectively. Electrolysis treatment produced very similar TCC concentrations, 8.8 × 104, 1.1 × 104 and 2.3 × 103 cells/mL. The TCC concentrations were lower than the concentration of the water entering each tank (∼3 × 105 cells/mL). Intact cell count (ICC) measurements indicated that the viable cell concentrations, were less than 10% of the TCC values. Though electrolysis treatment can produce powerful oxidants, such as hydroxyl radical, there was no evidence that electrolysis in this system provided additional benefits beyond chlorine production for control of total or intact cell counts. Oxidation of bacteria by chlorine was the dominant anti-bacterial mechanism in our system. Monitoring of dissolved organic carbon (DOC) and assimilable organic carbon (AOC) did not suggest that carbon-limitation significantly impacted cell counts when chlorination or electrolysis treatment was applied. This work demonstrates that either direct chlorination or electrolysis treatment are able to reduce bacteria concentrations over long-term operation of a hand washing water treatment system. We recommend selecting chlorine residual targets such that a chlorine residual is maintained during periods of challenging operating conditions. We observed that a target residual of 1 mg Cl2/L, in our system, maintained the TCC below the concentration found in Zurich drinking water.

Chemical composition, nutrient-balancing and biological treatment of hand washing greywater.

On-site biological hand washing water treatment can improve global access to safe hand washing water, but requires a thorough understanding of the chemical composition of the water to be treated, and an effective treatment strategy. This study first presents a detailed characterization of the individual inputs to hand washing water. We demonstrate (i) that soap is likely the most significant input in hand washing water, representing ∼90% of mass loading, and (ii) that inputs to hand washing water have low concentrations of biologically-essential macro- and micro-nutrients (nitrogen, phosphorus, potassium, copper, zinc, molybdenum and cobalt) with respect to carbon, which may impair biological carbon removal. This study next formulates a recipe that recreates a representative composition of hand washing water and develops a procedure to identify and supplement nutrients in which this recipe is estimated to be deficient. Batch testing of the nutrient-supplemented hand washing water with an inoculum of planktonic bacteria demonstrated improved assimilable organic carbon removal (99% vs. 86% removal) and produced lower final dissolved organic carbon concentrations (1.7 mgC/L vs. 3.5 mgC/L) compared to realistic (nutrient-deficient) washing water. Supplementing nutrients did promote cell growth (50x higher final total cell count). Full-scale testing in a biologically activated membrane bioreactor (BAMBi) system treating 75 L/day of nutrient-supplemented hand washing water showed that long-term operation (100 days) can deliver effective carbon removal (95%) without detrimental fouling or other disruptions caused by cell growth. This work demonstrates that biological treatment in a BAMBi system, operated with appropriate nutrient-balancing offers an effective solution for decentralized treatment of light greywater.

Controlling Bacterial Pathogens in Water for Reuse: Treatment Technologies for Water Recirculation in the Blue Diversion Autarky Toilet.

The Blue Diversion AUTARKY Toilet is a urine-diverting toilet with on-site treatment. The toilet is being developed to provide a safe and affordable sanitation technology for people who lack access to sewer-based sanitation. Water used for personal hygiene, hand washing, and flushing to rinse urine- and feces-collection bowls is treated, stored, and recycled for reuse to reduce reliance on external water supplies. The system provides an opportunity to investigate hygiene of water for reuse following treatment. Treatment in the toilet includes a Biologically Activated Membrane Bioreactor (BAMBi) followed by a secondary treatment technology. To identify effective secondary treatment, three options, including granular activated carbon (GAC) only, GAC+chlorine (sodium hypochlorite), and GAC+electrolysis are considered based on the bacterial inactivation and growth inhibition efficiency. Four different hygiene-relevant bacteria are tested: Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, and Salmonella typhimurium. Our evaluation demonstrates that-despite treatment of water with the BAMBi-E. coli, P aeruginosa, and S. typhimurium have the potential to grow during storage in the absence of microbial competition. Including the indigenous microbial community influences bacterial growth in different ways: E. coli growth decreases but P. aeruginosa growth increases relative to no competition. The addition of the secondary treatment options considerably improves water quality. A column of GAC after the BAMBi reduces E. coli growth potential by 2 log10, likely due to the reduction of carbon sources. Additional treatments including chlorination and electrolysis provide further safety margins, with more than 5 log-10 inactivation of E. coli. However, reactivation and/or regrowth of E. coli and P. aeruginosa occurs under in the absence of residual disinfectant. Treatment including the BAMBi, GAC, and electrolysis appear to be promising technologies to control bacterial growth during storage in water intended for reuse.