F-RNA Phages Research Articles

Assessing the prevalence of FRNA bacteriophages and their correlation with SARS-CoV-2 RNA in the wastewater of Mumbai city

ABSTRACT Bacteriophages have been explored as indicators of the presence of human viruses in water. F+ coliphages are of particular interest due to their abundance in wastewater and some groups surviving the treatment process. This study assessed the prevalence of F+ coliphages and FRNA bacteriophage (FRNAPH) groups in the wastewaters of Mumbai city and explored its correlation with the presence of SARS-CoV-2. Wastewater samples (raw = 63, treated = 99) collected from three wastewater treatment plants (WWTPs) were assessed for F+ coliphages, FRNAPH groups and SARS-CoV-2. Of the 63 raw wastewater samples, 92% were positive for F+ coliphages and FRNAPH, while SARS-CoV-2 was detected in 76% of samples; FRNAPH GI RNA was the most prevalent (96.82%) followed by FRNAPH GII (77.77%), FRNAPH GIII (36.50%), and FRNAPH GIV (4.76%). A significant correlation was observed between the occurrence of SARS-CoV-2 and F+ coliphages, FRNAPH GII and FRNAPH GIII RNA copies and also between F+ coliphages and FRNAPH GII and GIII. These observations suggest that F+ coliphages, FRNAPH GII and GIII can be explored further as indicators of SARS-CoV-2 in wastewaters. Also, F+ coliphage detection may be a cost-effective and practical approach for monitoring virus elimination during wastewater treatment.

Solid-liquid distribution of SARS-CoV-2 in primary effluent of a wastewater treatment plant

Distributions of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and fecal viral biomarkers between solid and liquid phases of wastewater are largely unknown. Herein, distributions of SARS-CoV-2, Pepper Mild Mottle Virus (PMMoV), and F-RNA bacteriophage group II (FRNAPH-II) were determined by viral RNA RT-qPCR. Comparison of viral recovery using three conventional fractionation methods included membrane filtration, a combination of mid-speed centrifugation and membrane filtration, and high-speed centrifugation. SARS-CoV-2 partitioned to the solids fraction in greater abundance compared to liquid fractions in a combination of mid-speed centrifugation and membrane filtration and high-speed centrifugation, but not in membrane filtration method in a particular assay, while fecal biomarkers (PMMoV and FRNAPH-II) exhibited the reciprocal relationship. The wastewater fractionation method had minimal effects on the solids-liquids distribution for all viral and phage markers tested; however, viral RNA load was significantly greater in solid–liquid fractions viral RNA loads compared with the than whole-wastewater PEG precipitation. A RNeasy PowerWater Kit with PCR inhibitor removal resulted in greater viral RNA loads and lesser PCR inhibition compared to a QIAamp Viral RNA Mini Kit without PCR inhibitor removal. These results support the development of improved methods and interpretation of WBE of SARS-CoV-2.•Distribution of SARS-CoV-2 to liquid and solid portions was addressed.•Addressing PCR inhibition is important in wastewater-based epidemiology.•Fraction methods have minimal effect.

Study of Shellfish Growing Area During Normal Harvesting Periods and Following Wastewater Overflows in an Urban Estuary With Complex Hydrography

Viral testing combined with hydrographic studies is considered standard good practice in determining microbiological impacts on shellfish growing areas following wastewater overflows. In this study, norovirus genogroup I and II, indicators of viral contamination (F-RNA bacteriophage genogroup II (F-RNA GII), crAssphage, pepper mild mottle virus) and Escherichia coli were monitored during periods of normal harvesting and following overflows in two commercial shellfish growing areas in Otago Harbour (Aotearoa New Zealand). Dye tracing, drogue tracking and analysis of particle tracking modelling were also undertaken to assess the dispersion, dilution and time of travel of wastewater discharged from a pump station discharge that impacts the growing areas. Norovirus was not detected in any of the 218 shellfish samples tested. PMMoV and crAssphage were more prevalent than F-RNA GII as determined by RT-qPCR. The dye study indicated long residence time of the waters (≥5 days) in the embayment impacted by the discharge. No relationships were found between the concentrations of viral indicators or E. coli and wastewater dilution, distance between the discharge and the growing areas or time since the last overflow. For the three spills studied (≤327 m3), there was little evidence of microbiological impact on the growing areas. This was likely associated with a deep shipping channel that enhances water flushing in the harbour and reduces contaminant transport to the growing areas. We recommend flexibility in the approach for closure/reopening growing areas impacted by spills, particularly for small duration/volume spills and when norovirus is not present in the community.

Laboratory Analysis of Passive Samplers Used for Wastewater-Based Epidemiology Using F-RNA Bacteriophage MS2 as a Model Organism

Laboratory Analysis of Passive Samplers Used for Wastewater-Based Epidemiology Using F-RNA Bacteriophage MS2 as a Model Organism

Applicability of F-specific bacteriophage subgroups, PMMoV and crAssphage as indicators of source specific fecal contamination and viral inactivation in rivers in Japan.

To date, several microbes have been proposed as potential source-specific indicators of fecal pollution. 16S ribosomal RNA gene markers of the Bacteroidales species are the most widely applied due to their predominance in the water environment and source specificity. F-specific bacteriophage (FPH) subgroups, especially FRNA phage genogroups, are also known as potential source-specific viral indicators. Since they can be quantified by both culture-based and molecular assays, they may also be useful as indicators for estimating viral inactivation in the environment. Pepper mild mottle virus (PMMoV) and crAssphage, which are frequently present in human feces, are also potentially useful as human-specific indicators of viral pollution. This study aimed to evaluate the applicability of FPH subgroups, PMMoV, and crAssphage as indicators of source-specific fecal contamination and viral inactivation using 108 surface water samples collected at five sites affected by municipal and pig farm wastewater. The host specificity of the FPH subgroups, PMMoV, and crAssphage was evaluated by principal component analysis (PCA) along with other microbial indicators, such as 16S ribosomal RNA gene markers of the Bacteroidales species. The viabilities (infectivity indices) of FRNA phage genogroups were estimated by comparing their numbers determined by infectivity-based and molecular assays. The PCA explained 58.2% of the total information and classified microbes into three groups: those considered to be associated with pig and human fecal contamination and others. Infective and gene of genogroup IV (GIV)-FRNA phage were assumed to be specific to pig fecal contamination, while the genes of GII-FRNA phage and crAssphage were identified to be specific to human fecal contamination. However, PMMoV, infective GI-FRNA phage, and FDNA phage were suggested to not be specific to human or pig fecal contamination. FRNA phage genogroups, especially the GIV-FRNA phage, were highly inactivated in the warm months in Japan (i.e., July to November). Comparing the infectivity index of several FRNA phage genogroups or other viruses may provide further insight into viral inactivation in the natural environment and by water treatments.

Rainwater for residential hot water supply: Managing microbial risks

There is growing interest in using alternative water sources such as rainwater harvesting and treatment systems to supplement traditional sources and secure a stable supply. For such systems, it is important to ensure adequate water quality, as microbial contamination can be a risk factor in rainwater. The primary objective of this study was to provide proof-of-concept for the microbial treatment capacity of a residential scale rain-to-hot-water treatment system to be installed in Melbourne, Australia. The system consists of a filtration, UV and heat-pump hot water unit, and disinfects roofwater prior to use of the hot water for bath, shower and laundry. The system's efficiency was evaluated using long-term challenge tests investigating the full system and each of the separate components. The microbial treatment performance was assessed based on the systems' ability to treat high levels of E. coli, E. faecalis, Campylobacter, Salmonella and MS2 phage under challenging conditions; with varying flow rates (10-40 L/min) and microbe concentrations (104-105unit/L), and in scenarios of a power outage. Over a compressed year of operation, the full rain-to-hot-water treatment system was extremely efficient at reducing concentrations of E. coli, E. faecalis, Campylobacter, Salmonella and FRNA phages, with log reductions ranging from the lowest average of 2.1log reductions for Salmonella to a maximum of >5.1log for E. coli. Most of the treatment was provided by the UV system, and any remaining microbes present after this point were deactivated by the heat-pump system (provided that the water was given enough time to warm up). Additional modelling work showed that UV intensity, UV transmissivity and contact time (for the UV system) and temperature (for the heat-pump hot water system) could be used as parameters to predict microbial treatment performance of the system, indicating that these easily-measurable parameters could assist with ongoing operation optimisation and maintenance of such systems.

Application of crAssphage, F-RNA phage and pepper mild mottle virus as indicators of human faecal and norovirus contamination in shellfish

Shellfish growing waters contaminated with inadequately treated human wastewater is a major source of norovirus in shellfish and poses a significant human health risk to consumers. Microbial source tracking (MST) markers have been widely used to identify the source (s) of faecal contamination in water but data are limited on their use for shellfish safety. This study evaluated the source specificity, sensitivity, occurrence and concentration of three viral MST markers i.e. cross-assembly phage (crAssphage), F-specific RNA bacteriophage genogroup II (F-RNA phage GII) and pepper mild mottle virus (PMMoV) using animal faeces (n = 119; 16 animal groups), influent wastewater (n = 12), effluent wastewater (n = 16) and shellfish (n = 33). CrAssphage, F-RNA phage GII and PMMoV had source specific values of 0.97, 0.99 and 0.91, respectively. The sensitivity of MST markers was confirmed by their 100% detection frequency in influent wastewaters. The frequency of detection in effluent wastewater ranged from 81.3% (F-RNA phage GII) to 100% (PMMoV). Concentration of F-RNA phage GII was one log10 (influent wastewater) and 2–3 log10 (effluent wastewater) lower than crAssphage and PMMoV, respectively. Despite lower prevalence of F-RNA phage GII in oysters and mussels compared to crAssphage and PMMoV, concentrations of the three MST markers were similar in mussels. As an indicator of norovirus contamination in shellfish, crAssphage and PMMoV had greater predictive sensitivity (100%; [95% CI; 81.5%–100%)]) and F-RNA phage GII had greater predictive specificity (93.3%; [95% CI; 68.1%–99.8%]). In contrast, crAssphage and F-RNA phage GII have similar accuracy for predicting norovirus in shellfish, however, PMMoV significantly overestimated its presence. Therefore, a combination of crAssphage and F-RNA phage GII analysis of shellfish could provide a robust estimation of the presence of human faecal and norovirus contamination.

Comparison of faecal indicator and viral pathogen light and dark disinfection mechanisms in wastewater treatment pond mesocosms

This study compared light and dark disinfection of faecal bacteria/viral indicator organisms (E. coli and MS2 (fRNA) bacteriophage) and human viruses (Echovirus and Norovirus) in Wastewater Treatment Pond (WTP) mesocosms. Stirred pond mesocosms were operated in either outdoor sunlight-exposed or laboratory dark conditions in two experiments during the austral summer. To investigate wavelength-dependence of sunlight disinfection, three optical filters were used: (1) polyethylene film (light control: transmitting all solar UV and visible wavelengths), (2) acrylic (removing most UVB <315 nm), and (3) polycarbonate (removing both UVB and UVA <400 nm). To assess different dark disinfection processes WTP effluent was treated before spiking with target microbes, by (a) 0.22 μm filtration to remove all but colloidal particles, (b) 0.22 μm filtration followed by heat treatment to destroy enzymes, and (c) addition of Cytochalasin B to supress protozoan grazing. Microbiological stocks containing E. coli, MS2 phage, Echovirus, and Norovirus were spiked into each mesocosm 10 min before the experiments commenced. The light control exposed to all sunlight wavelengths achieved >5-log E. coli and MS2 phage removal (from ~1.0 × 106 to <1 PFU/mL) within 3 h compared with up to 6 h in UV-filtered mesocosms. This result confirms that UVB contributes to inactivation of E. coli and viruses by direct sunlight inactivation. However, the very high attenuation with depth of UVB in WTP water (99% removal in the top 8 cm) suggests that UVB disinfection may be less important than other removal processes averaged over time and full-scale pond depth. Dark removal was appreciably slower than sunlight-mediated inactivation. The dark control typically achieved higher removal of E. coli and viruses than the 0.22 μm filtered (dark) mesocosms. This result suggests that adsorption of E. coli and viruses to WTP particles (e.g., algae and bacteria bio-flocs) is an important mechanism of dark disinfection, while bacteria and virus characteristics (e.g. surface charge) and environmental conditions can influence dark disinfection processes.

Investigation of F-RNA Bacteriophage as a Tool in Re-Opening Australian Oyster Growing Areas Following Sewage Spills.

Oysters contaminated with human enteric viruses from sewage are implicated in foodborne outbreaks globally. Bacteriophages have been identified as potential indicators for these viruses, but have not been used in shellfish management outside of the USA. This study aimed to determine the background levels of F-RNA phage in five Australian oyster growing areas with a history of sewage spills and closures, over an 18-month period. In addition, oysters from five growing areas impacted by adverse sewage events were investigated for F-RNA phage, Escherichia coli, norovirus (NoV) and hepatitis A virus (HAV). F-RNA phage ≤ 60 pfu/100 gm shellfish flesh were found to represent a conservative background level in the surveyed areas. Following two of the five sewage spills, elevated phage levels were observed in most sample sites less than 4days post spill. By 7days, most sites from all events had phage < 30 pfu/100 gm. NoV was detected in day 1 and day 6 samples from one event when all phage were ≤ 30 pfu/100 gm. NoV was also detected in a day 3 sample from another event with < 30 phage pfu/100 gm, however, multiple replicate samples had elevated phage levels. The results of this study add evidence on the potential use of F-RNA phage as a tool in early re-opening of oyster harvest areas post sewage spills. However, it also highlights the need to better understand situations where phage testing may be ineffectual, and the importance of sampling at multiple sites and over multiple time points, to effectively capture evidence of contamination.

Effectiveness and Energy Requirements of Pasteurisation for the Treatment of Unfiltered Secondary Effluent from a Municipal Wastewater Treatment Plant

Pasteurisation was investigated as a process to achieve high microbial quality standards in the recycling of water from unfiltered secondary effluents from a wastewater treatment plants in Melbourne, Australia. The relative heat sensitivity of key bacterial, viral, protozoan and helminth wastewater organisms (Escherichia coli, Enterococcus, FRNA bacteriophage, adenovirus, coxsackievirus, Cryptosporidium, and Ascaris) were determined by laboratory scale tests. The FRNA phage were found to be the most heat resistant, followed by enterococci and E. coli. Pilot scale challenge testing of a 2 ML/day pasteurisation pilot plant using unfiltered municipal wastewater and male specific coliphage (MS2) phage showed that temperatures between 69 °C and 75 °C achieved log reductions values between 0.9 ± 0.1 and 5.0 ± 0.5 respectively in the contact chamber. Fouling of the heat exchangers during operation using unfiltered secondary treated effluent was found to increase the energy consumption of the plant from 2.2 kWh/kL to 5.1 kWh/kL. The economic feasibility of pasteurisation for the current municipal application with high heat exchanger fouling potential can be expected to depend largely on the available waste heat from co-generation and on the efforts required to control fouling of the heat exchangers.

Spatial and temporal profiles of enteric viruses in the coastal waters of Tokyo Bay during and after a series of rainfall events

Recreational activities in coastal waters that are polluted by enteric viruses can result in gastroenteritis etc. In this study, the pollution profiles of enteric viruses were examined in the coastal area of Tokyo Bay, Japan, by collecting 57 water samples from three different depths (0.5 m, 3.0 m, and 5.0 m) during and after a series of heavy rainfall events. Vertically spatial and temporal changes in the concentrations of NoV genogroup I (GI) and genogroup II (GII), pepper mild mottle virus (PMMoV), and Aichi virus (AiV) were determined using quantitative reverse transcription-polymerase chain reaction, while those of the bacterial indicator, Escherichia coli, and F-specific RNA bacteriophages (FRNA phages) were monitored using culture methods. PMMoV was highly abundant (1.4 × 104–6.8 × 106 genome copies/L), whereas the concentrations of the other enteric viruses were relatively low (AiV, 1.3 × 102–2.9 × 104; GI, 2.9 × 10–5.6 × 103; GII, 2.5 × 10–1.2 × 104 genome copies/L). All of the viruses showed lower fluctuations in concentration than E. coli, which increased up to 460-fold after the rainfall event and then decreased over the subsequent two weeks. The maximum vertical difference in E. coli concentration was observed immediately after the rainfall. The E. coli reached the surface and then gradually spread down, whereas the virus concentrations exhibited few fluctuations due to the remaining effects of the previous combined sewer overflows. These findings indicate that viruses have a relatively long retention period over fecal indicator bacteria in this coastal area.

Microbial pathogen removal from domestic effluent using coral sand in Kiribati.

Infiltration experiments aimed to show the effectivity of coral sand to remove micro-organisms in septic tank wastewater treatment system, in South Tarawa, Kiribati. Laboratory experiments evaluated effective microbial removal properties of a packed bed of coral beach sand conditioned with domestic effluent. Bacterial and viral indicators Escherichia coli J6-2, Enterococci faecalis and f-RNA (MS2) bacteriophage, along with viral pathogens adenovirus, echovirus, norovirus and rotavirus, were dosed (at 106 -107 CFU, PFU or genome copies per ml concentration) with effluent to unsaturated coral sand-packed columns. For the conditions simulated, all organisms showed removal efficiencies of >4-Log Removal Values (LRVs) (i.e. >99·99% effective reduction in number). Results revealed that the conditioned coral sand had a higher affinity for attenuating viruses than the bacteria tested. MS2 phage, adenovirus, echovirus, norovirus and rotavirus were absent in leachate from the base of the sand columns. E. coli J6-2 and E. faecalis were recovered at 100 -101 CFU (colony forming units) per ml in the column effluent, following a dosage of 106 CFU per ml. Destructive sampling of the columns after experiments revealed that a high proportion of viral pathogens were retained in the top 30mm of the sand. On the basis of the relative spatial distribution of trace organisms in the sand, we speculate that adsorption processes likely dominated attenuation of the viral pathogens, more so than physical straining effects. Further study is required to fully elucidate the removal mechanisms. To the best of our knowledge this is the first study of the effective microbial removal capacity of coral sand under unsaturated conditions. The work represents an initial exploratory step of developing some standardized design practice of on-site wastewater treatment systems in Kiribati, to offer enhanced protection of groundwater resources and reduce diarrhoeal disease.

Development of a Portable Detection Method for Enteric Viruses from Ambient Air and Its Application to a Wastewater Treatment Plant.

The ambient air from wastewater treatment plants has been considered as a potential source of pathogenic microorganisms to cause an occupational risk for the workers of the plants. Existing detection methods for enteric viruses from the air using a liquid as the collection medium therefore require special care to handle on-site. Knowledge accumulation on airborne virus risks from wastewater has been hindered by a lack of portable and handy collection methods. Enteric viruses are prevalent at high concentrations in wastewater; thus, the surrounding air may also be a potential source of viral transmission. We developed a portable collection and detection method for enteric viruses from ambient air and applied it to an actual wastewater treatment plant in Japan. Materials of the collection medium and eluting methods were optimized for real-time polymerase chain reaction-based virus quantification. The method uses a 4 L/min active air sampler, which is capable of testing 0.7–1.6 m3 air after 3–7 h sampling with a detection limit of 102 copies/m3 air in the field. Among 16 samples collected at five to seven locations in three sampling trials (November 2007–January 2008), 56% (9/16) samples were positive for norovirus (NV) GII, with the highest concentration of 3.2 × 103 copies/m3 air observed at the sampling point near a grit chamber. Adenoviruses (4/16), NV GI (6/16), FRNA bacteriophages GIII (3/16), and enteroviruses (3/16) were also detected but at lower concentrations. The virus concentration in the air was associated with that of the wastewater at each process. The results imply that the air from the sewer pipes or treatment process is contaminated by enteric viruses and thus special attention is needed to avoid accidental ingestion of viruses via air.

Bacteria and virus removal in denitrifying bioreactors: Effects of media type and age

Denitrifying bioreactors are simple, low-cost ecotechnologies designed to reduce nitrate (NO3−) present in septic tank effluent and drainage water. Recent studies indicate that, in addition to significant reduction in NO3− loads, these systems are also able to remove microbial contaminants from municipal wastewater. However, the removal of microbial contaminants in denitrifying bioreactors remains poorly characterised and factors that control removal in denitrifying bioreactors remain unexplored. In this study, the removal efficiency of faecal indicator bacteria Escherichia coli (E. coli) and total coliforms (TC) as a model for bacterial pathogens and F-specific RNA bacteriophage (FRNA bacteriophage) as a model for viruses was assessed for mesocosm-scale (∼700 L) bioreactors receiving municipal wastewater. Systems were filled with two different slow-release carbon sources: woodchip and coconut husk. The effect of media age on attenuation of microbial contaminants was assessed by comparing the performance of 8-year old systems with equivalent newly constructed woodchip and coconut husk bioreactors. Additionally, removal performance of these carbon substrates was compared to that of gravel, a non-carbon substrate commonly used in subsurface flow (SSF) constructed wetlands. Substantial reduction of E. coli, TC and FRNA bacteriophage from primary treated municipal wastewater was achieved in all bioreactors. Mean annual log10 removal efficiencies were similar between microbial indicators ranging from 1.4 to 1.9 for TC, 1.3 to 1.8 for E. coli and 1.3 to 2.0 for FRNA bacteriophage. All denitrifying bioreactors showed consistent year-round performance and long-term performance which did not markedly change in the ninth year of operation. The woodchip or coconut husk bioreactors achieved microbial effluent quality within the same range of log10 removal rates achieved in gravel-based systems. This suggests that denitrifying bioreactors, as well as reducing N loads, can effectively reduce microbial contaminants in wastewater, providing a complimentary disinfection role. Further research is needed to increase understanding of factors affecting removal of microbial contaminants in denitrifying bioreactors to support design of these systems for microbial contaminant removal.

Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100copies/g norovirus and > 10pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.

Spatio-temporal dynamics of virus and bacteria removal in dual-media contact-filtration for drinking water

Microorganism removal efficiencies in deep bed filters vary with time and depth in the filter bed as the filter collects particles. Improved knowledge of such dynamics is relevant for the design, operation and microbial risk assessment of filtration processes for drinking water treatment. Here we report on a high-resolution spatio-temporal characterization of virus and bacteria removal in a pilot-scale dual-media filter, operated in contact-filtration mode. Microorganisms investigated were bacteriophage Salmonella typhimurium 28B (plaque assay, n=154)), fRNA phage MS2 (plaque assay/RT-qPCR, n=87) and E. coli (Colilert-18, n=73). Microscopic and macroscopic filtration models were used to investigate and characterize the removal dynamics.Results show that ripening/breakthrough fronts for turbidity, viruses and E. coli migrated in a wave-like manner across the depth of the filter. Virus removal improved continuously throughout the filter cycle and viruses broke through almost simultaneously with turbidity. Ripening for E. coli took longer than ripening for turbidity, but the bacteria broke through before turbidity breakthrough. Instantaneous log-removal peaked at 3.2, 3.0 and 4.5 for 28B, MS2 and E. coli, respectively. However, true average log-removal during the period of stable effluent turbidity was significantly lower at 2.5, 2.3 and 3.6, respectively. Peak observed filter coefficients λ were higher than predicted by ideal filtration theory. This study demonstrates the importance of carefully designed sampling regimes when characterizing microorganism removal efficiencies of deep bed filters.

Comparing the performance of aerobic granular sludge versus conventional activated sludge for microbial log removal and effluent quality: Implications for water reuse

The application of aerobic granular sludge (AGS) technology has increased in popularity, largely due to the smaller physical footprint, enhanced biological nutrient removal performance and ability to perform with a more stable operation when compared to conventional activated sludge (CAS) systems. To date, the ability of AGS to remove microbial pathogens such as; Escherichia coli, Giardia, and Cryptosporidium has not been reported. This study compared the log10 removal performance of commonly used pathogen surrogates (sulfite-reducing clostridia spores, f-RNA bacteriophage, E. coli and total coliforms) by AGS and CAS during the start-up phase, through to maturation. Results showed that AGS performed as well as CAS for the log10 removal performance of all microbial surrogates, except for spores which were removed more effectively by AGS most likely due to greater adherence of spores to the AGS biomass compared to CAS mixed liquor. Results suggest that AGS is capable of meeting or exceeding CAS-equivalent health-based targets for pathogen removal in the context of water recycling as well as not adversely affecting the secondary effluent water quality (suspended solids, turbidity and particle size) in terms of ultraviolet light transmissivity (254 nm). These findings confirmed for the first time that the adoption of AGS operation would not adversely impact downstream tertiary disinfection processes from altered water quality, nor would it require further pathogen treatment interventions in addition to what is already required for CAS systems.

Pond walls: inclined planes to improve pathogen removal in pond systems for wastewater treatment?

Attenuation of sunlight in wastewater treatment ponds reduces the depth of the water exposed to disinfecting irradiances. Shallow pond depth with paddlewheel rotation increases exposure of pathogens to sunlight in high rate algal ponds. Generation of thin films, using pond walls as inclined planes, may increase inactivation of pathogens by increasing sunlight exposure. The performance of a laboratory based model system incorporating an inclined plane (IP) was evaluated. F-RNA bacteriophage, in tap water or wastewater, was exposed to sunlight only on the IP with the bulk water incubated in the dark. MS2 inactivation was significantly higher when the IP was present (P < 0.05) with a 63% increase observed. Prolonged exposure increased MS2 die-off irrespective of IP presence. Versatility of the IP was also demonstrated with faster inactivation observed in both optically clear tap water and wastewaters. IPs of different surface areas produced similar inactivation rates when operated at similar hydraulic loading rates regardless of slope length.

Decentralised black water treatment by combined auto-thermal aerobic digestion and ammonia – A pilot study optimising treatment capacity

Partial heating of black water by auto-thermal aerobic digestion was combined with the addition of 1% w/w urea and monitoring of pathogens and indicator organisms over a 21-day period. After initial mixing, the 160 m3 black water (60 m3 heated and 100 m3 non-heated) was left undisturbed. The urea was confirmed to be fully degraded into ammonia (5.1 g N L−1) first after 14 days, while the pH stabilised at around 9.2 after one week. The initial temperature of 17 °C fell by 6 °C during the study. E. coli and Salmonella spp., which are sensitive to ammonia, were inactivated during the first few days of the study, despite the urea only being partly hydrolysed. At day 14, f-RNA bacteriophages could also no longer be detected. The more persistent somatic coliphages, Enterococcus spp. and Ascaris eggs, showed significant but slow inactivation. The treatment proved to be efficient with regards to salmonella, which is a target pathogen in the Swedish context, but for parasite egg inactivation a higher temperature was required. The treatment would benefit from more frequent stirring to speed up the hydrolysis of urea and thus improve treatment efficiency. The alternative treatment scheme could increase capacity by 2.4 times, albeit with a 40% higher cost per volume due to the increased use of urea.

DISTRIBUTION OF FECAL INDICATOR BACTERIA IN SEDIMENT OF LOCAL OPEN CHANNELS RECEIVING JOHKASOU EFFLUENT

Onsite wastewater treatment system named Johkasou has been widely applied in the Japanese rural areas, which the effluent is mainly discharged into stream channels. However, Johkasou effluent would potentially be a source of contamination that can deteriorate water and sediment quality of the receiving downstream network. In order to identify the effect of Johkasou effluent particularly in sediment, a study on the distribution of fecal indicators (F-RNA bacteriophages, total coliform (TC), and Escherichia coli [E. coli]) in several sediment spots of local open channels receiving Johkasou effluent was conducted. The results showed that the contents of F-RNA bacteriophages, TC and E. coli in the sediment of open channels were detected in high levels while those contents in the sediment of Johkasou drainage channel were up to two orders magnitude higher than in the open channel. A high number of fecal bacteria in the receiving open channels was majorly influenced by the number of domestic households. Moderate correlations of fecal bacteria indicator with solid sediment and volatile sediment indicate that the sediment particles can be carriers of microbes to the downstream water networks. Therefore, water flushing and hydraulic events are suggested to reduce sediment depth that contained fecal bacteria indicator in the open channel.