Removal of coliphages in secondary effluent by microfiltration-mechanisms of removal and impact of operating parameters

The efficacy of a microfiltration (MF) pilot plant in removing somatic coliphages (referred hereafter as coliphages) present in the secondary effluent was evaluated during this study. The impact of operating parameters such as feed coliphage concentrations, permeate flux and membrane fouling on the removal of coliphages by the MF plant was investigated. The study showed that membrane fouling was beneficial for removing coliphages by MF. It was also shown that the removal of coliphages by MF was initially governed by adsorption on membrane surface or in membrane pores. As the membrane fouled, however, the removal of coliphages was primarily governed by direct interception on the cake layer formed on the surface of the membrane. Increases in feed coliphage concentrations resulted in the passage of larger numbers of coliphages when the MF was clean but had little impact on the passage of coliphages when the membrane became fouled. Increasing permeate flux lowered log-removal values (LRVs) for the clean membrane but resulted in an initial increase in LRVs for the fouled membrane followed by a drop in LRVs with further increases in permeate flux.     

Viruses and bacteriophages

Many of the enteric viruses which are transmitted from person to person by thefecal-oral route are found in raw and treated wastewater, and because of their persistence under adverse conditions may also be found in slightly polluted waters.There is no routine examination procedure of water and wastewater for entero-viruses,mainly because of the cumbersome isolation techniques, high cost and the need for highly skilled laboratory personnel.Phages are specific to single species of bacteria, are known for many entericbacteria, and are very often used for final identification of enteric pathogenic bacteria.Coliphages are provalent in raw and treated sewage as well as in polluted water, where enteric viruses may also be found.Coliphages were often mentioned as possible viral indicators in polluted water.To be a perfect indicator, they should comply with minimum criteria as follows: (a) they should be present wherever human enteric viruses are present: (b) the coliphage numbers recovered should be equal to or larger than those of enteric viruses recovered: (c) the coliphages should be at least as resistant as enteric viruses to adverse environmental conditions: (d) isolation and quantification of the coliphage should be faster and less expensive than isolation of the enteroviruses.Comparative studies show that the coliphage to enterovirus ratio in wastewater is about 10³:1. Levels of poliovirus 1 (attenuated) to coliphage f2 remained stable for a few months in oxidation pond effluents.f2 coliphage exhibited higher resistance to chlorination than poliovirus 1 (attenuated). When the two strains were kept in water of different quality, f2 survived longer. In addition, all coliphage counts were completed within 24 h. while those of enteroviruses required about a week. Results indicate very strongly that coliphages can be used as viral indicators and this is already the practice in a few European and other countries.     

Removal of native coliphages and coliform bacteria from municipal wastewater by various wastewater treatment processes: implications to water reuse

The efficacy of a conventional activated sludge wastewater treatment process and the membrane bioreactor technology in removing microbial pathogens was investigated. Total and fecal coliforms and somatic and F-specific coliphages were used as indicators of pathogenic bacteria and viruses. Up to 5.7 logs removal of coliforms and 5.5 logs of coliphages were observed in the conventional treatment process with advanced tertiary treatment. Addition of chemical coagulants seemed to improve the efficacy of primary and secondary treatment for microorganism removal. Complete removal of fecal coliforms and up to 5.8 logs removal of coliphages was observed in the MBR system. It was shown that the MBR system was capable of high removal of coliphages despite the variation in feed coliphage concentrations. The results of this study indicated that the MBR system can achieve better microbial removal in far fewer steps than the conventional activated sludge process with advanced tertiary treatment. The final effluent from either treatment processes can be potentially reused.     

Survival of prototype strains of somatic coliphage families in environmental waters and when exposed to UV low-pressure monochromatic radiation or heat

The potential use of specific somatic coliphage taxonomic groups as viral indicators based on their persistence and prevalence in water was investigated. Representative type strains of the 4 major somatic coliphage taxonomic groups were seeded into reagent water and an ambient surface water source of drinking water and the survival of the added phages was measured over 90 days at temperatures of 23-25 and 4 °C. Microviridae (type strain PhiX174), Siphoviridae (type strain Lambda), and Myoviridae (type strain T4) viruses were the most persistent in water at the temperatures tested. The Microviridae (type strain PhiX174) and the Siphoviridae (type strain Lambda) were the most resistant viruses to UV radiation and the Myoviridae (type strain T4) and the Microviridae (type strain PhiX174) were the most resistant viruses to heat. Based on their greater persistence in water over time and their relative resistance to heat and/or UV radiation, the Myoviridae (type strain T4), the Microviridae (type strain PhiX174), and the Siphoviridae (type strain Lambda) were the preferred candidate somatic coliphages as fecal indicator viruses in water, with the Microviridae (type strain PhiX174) the most resistant to these conditions overall.     

F-specific RNA coliphages: occurrence,types, and survival in natural waters

A small, well-defined watershed was investigated over a 2-year period to determine the prevalence of F-specific RNA coliphage (F + RNA) serotypes as indicators of animal fecal contamination. Sampling sites collected runoff from areas of urban and agricultural land use patterns. F-specific coliphages were concentrated from 2-L freshwater samples by polyethylene glycol precipitation, isolated using the double agar layer (DAL) method, confirmed as F + RNA by RNAse suppression, and serotyped. A subset of serotyped F + RNA were confirmed by genotyping. To determine relative survival, 10 confirmed F + RNA field isolates and 5 prototypic F + RNA were spiked into surface water and incubated at 25 degrees C for 36 days. F-specific coliphage isolation was strongly associated with rainfall events and was infrequent from primarily animal impacted surface waters. Field isolates were predoffiinantly Type I F + RNA (81%) and raw sewage isolates were predominantly Type III F + RNA (57%). Genotyping from either the watershed or raw sewage samples never positively identified Type IV F + RNA. Results from laboratory studies showed that F + RNA differ in their survival in water and that Type IV strains were the least persistent. Type III F + RNA were found to be reliably related to the release of uncontrolled human fecal material in the watershed, but the results of this study suggest that further study is required before utilizing for fecal source identification in natural waters.     

Coliphage prevalence in high school septic effluent and associated ground water

At the present time, somatic and male-specific coliphage and human enterovirus groups are being considered as indicators of possible pathogenic human enteric virus contamination from fecal contamination. A primary attribute for any indicator of fecal contamination is its prevalence at the source and in associated ground water. It must be consistently found in the source material at concentrations that are measurable with available techniques. Over a period of ten months, male-specific and somatic coliphage ranged from 7000 to 4,000,000 PFU/L in the effluent from a multi-user septic-tank. Unlike the values determined for septic-tank effluent, coliphage concentrations measured in ground water over this same period only varied by five-fold. Coliphage concentration in ground water under the down-gradient edge of the drainfield contained 1000 PFU/L. This concentration decreased at −1 log₁₀/5 m during 17.4 m of ground-water transport. From these data, coliphage concentrations in septic-tank effluent seem sufficient to allow their use as indicators of fecal contamination in ground water.     

The limitation of the ratio of fecal coliforms to total coliphage as a water pollution index

The fecal coliform populations of raw sewage, sewage lagoon effluent, and river water were determined using the most probable number technique. The total coliphage populations of the three water sources were determined using Escherichia coli B (ATCC 11303-1) host cells. The ratios of fecal coliforms to coliphage in the three water samples were 87:1, 4.2:1, and 0.15:1, respectively. The ratio of fecal coliforms to coliphage in stored raw sewage decreased from 87:1 to about 1:1 within 7 days at 20°C and within 28 days at 4°C. These changing ratios resulted from the greater longevity of the coliphage compared with that of the coliform bacteria. The use of the ratio of fecal coliforms to coliphage is not considered reliable as an index of when a fecal pollution event occurred because the ratio is influenced by prior contamination, presence of sediment, chlorination, and temperature.     

Coliphages as indicators of enteric viruses in activated sludge

The use of indigenous coliphage as indicators of enteric viruses during activated sludge treatment of domestic wastewater was examined. Enteric viruses, coliphage attacking three strains of Escherichia coli and a number of wastewater parameters were monitored in the primary effluent, secondary effluent and secondary sludge of a wastewater treatment plant.Removal of viruses during secondary treatment appeared to be the result of rapid adsorption of influent virions to mixed liquor suspended solids. Adsorption was followed by inactivation of the viruses during aeration of the mixed liquor. A larger proportion of influent enteric viruses than coliphage was recovered from the secondary sludge. This suggests that activated sludge treatment was less antagonistic towards enteric viruses than towards the coliphage.Coliphage plaques of less than or equal to 1 mm in diameter were composed of large viruses (approx. 100 nm in diameter). Plaques greater than 3 mm in diameter appeared to be the result of host infection by a much smaller virus (approx. 45 nm in diameter). Plaques between 1 and 3 mm in diameter were composed of both small and large coliphages.Coliphage concentrations could not be correlated with enteric virus concentrations in either primary or secondary effluent. However, it was found that coliphage giving rise to plaques greater than 3 mm in diameter were positively related with enteric viruses in the secondary effluent. This result suggests that this group of coliphage may serve as an indicator of the efficacy of activated sludge treatment of enteric viruses.     

Microorganism removal from wastewater by rapid mixed media filtration

Reuse of domestic sewage effluents in Arizona requires that certain microbiological standards be met. The purpose of this project was to determine the effectiveness of rapid mixed media filtration in the removal of enteric viruses, fecal coliforms and roundworm eggs from sewage destined for reuse as irrigation water. Two surrogate viruses, the simian rotavirus SA-11, used in place of human rotavirus which is pathogenic to man, and f2 coliphage, known to adsorb poorly to surfaces, were used to evaluate filter performance. No significant removal of coliphages occurred by the filters. Addition of ferric chloride and anionic polymer did not enhance virus removal. When small amounts of alum and polymer were added, removal of colliphage increased to 37–40%. Average removal of rotavirus ranged from 21 to 27% after addition of alum and polymer. Mixed media filtration effectively reduced coliform numbers. The removal of Ascaris ova by filtration was essentially complete.     

Survival of infectious Poliovirus-1 in river water compared to the persistence of somatic coliphages, thermotolerant coliforms and Poliovirus-1 genome

The microbiological quality of water is currently assessed by search for fecal bacteria indicators. There is, however, a body of knowledge demonstrating that bacterial indicators are less resistant to environmental factors than human pathogenic viruses and therefore underestimate the viral risk. As river water is often used as a resource for drinking water production, it is particularly important to obtain a valid estimation of the health hazard, including specific viral risk. This work was conducted to compare the survival of infectious Poliovirus-1 used as a pathogenic virus model to the persistence of, on the one hand, thermotolerant coliforms commonly used as indicators and on the other hand, to somatic coliphages and Poliovirus-1 genome considered as potential indicators. We studied the behavior of infectious Poliovirus-1 and the three (potential) indicators of viral contamination in river water at three different temperatures (4 degrees C,18 degrees C and 25 degrees C). This experiment was performed twice with river water sampled at two different periods, once in winter and once in summer. Our results showed that the survival of thermotolerant coliforms can be 1.5-fold lower than infectious Poliovirus-1. In contrast, under all our experimental conditions, somatic coliphages and Poliovirus-1 genome persisted longer than infectious Poliovirus-1, surviving, respectively, 2-6-fold and about 2-fold longer than infectious Poliovirus-1. According to our results exclusively based on survival capacity, somatic coliphages and viral genome, unlike thermotolerant coliforms appear to be better indicators of viral contamination in river water. Moreover, the disappearance of viral genome is well-correlated to that one of infectious virus irrespective of the conditions tested.     

Modeling Enterococcus densities measured by quantitative polymerase chain reaction and membrane filtration using environmental conditions at four Great Lakes beaches

Data collected by the US Environmental Protection Agency (EPA) during the summer months of 2003 and 2004 at four US Great Lakes beaches were analyzed using linear regression analysis to identify relationships between meteorological, physical water characteristics, and beach characteristics data and the fecal indicator bacteria, Enterococcus. Water samples were analyzed for Enterococcus densities by quantitative polymerase chain reaction (qPCR) and membrane filtration (MF). This paper investigates the ability of regression models to accurately predict Enterococcus densities above or below a threshold value, using environmental data on a beach-by-beach basis for both methods. The ability to create statistical models for real-time water quality analysis would allow beach managers to make more accurate decisions regarding beach safety. Results from linear regression models indicate that environmental factors explain more of the variability in Enterococcus densities measured by MF than Enterococcus densities measured by qPCR. Results also show that models for both methods did not perform well at predicting occurrences in which water quality levels exceeded a threshold.     

Peak flux performance and microbial removal by selected membrane bioreactor systems

A pilot study was conducted over a period of 18 months at the Point Loma Wastewater Treatment Plant (PLWWTP) in San Diego, CA to evaluate the operational and water quality performance of six selected membrane bioreactor (MBR) systems at average and peak flux operation. Each of these systems was operated at peak flux for 4 h a day for six consecutive days to assess peak flux performance. Virus seeding studies were also conducted during peak flux operation to assess the capability of these systems to reject MS-2 coliphage. When operating at steady state, these MBR systems achieved an effluent BOD concentration of <2 mg/L and a turbidity of <0.1 NTU. Peak flux for the MBR systems ranged from 56 to 76 L/m²/h (liters per square meter per hour) with peaking factors in the range of 1.5-3.2. When switching from average to peak flux operation, a reversible drop of 22-32% in temperature-corrected permeability was observed for all submerged MBR systems. The percent drop in permeability increased as MLSS concentration in the membrane tank increased from 11,100 mg/L to 15,300 mg/L and was observed to be highest for the system operating at highest MLSS concentration. Such trends were not observed with an external MBR system. Each MBR system was able to sustain a 4-h-a-day peak flow for six consecutive days with only moderate membrane fouling. The membrane fouling was quantified by measuring the drop in temperature-corrected permeability. This drop ranged from 13 to 33% over six days for different submerged MBR systems. The MBR systems achieved microbial removal in the range of 5.8-6.9 logs for total coliform bacteria, >5.5 to >6.0 logs for fecal coliform bacteria and 2.6 to >3.4 logs for indigenous MS-2 coliphages. When operating at peak flux, seeded MS-2 coliphage removal ranged from 1.0 to 4.4 logs, respectively. The higher log removal values (LRVs) for indigenous MS-2 coliphage among different MBR systems were probably the result of particle association of indigenous coliphage. Differences in membrane pore size (0.04-0.2 μm) amongst the MBR systems evaluated did not have a substantial impact on indigenous MS-2 coliphage removal, but seeded MS-2 coliphage removal varied among the different MBR systems.     

Investigation of norovirus occurrence in groundwater in metropolitan Seoul, Korea

Groundwater is an important source of drinking and household water worldwide. Hence, the quality of groundwater is very important for preventing waterborne disease outbreaks and should be properly monitored. This study investigated the prevalence of waterborne viruses and fecal indicators in groundwater in metropolitan Seoul and Gyeonggi province, South Korea. A total of 116 samples of groundwater were taken using NanoCeram filters during both summer (June to August) and fall-winter seasons (October to December) in 2008. Among 71 sampling sites, 28 (48.3%) and 18 (35.3%) were positive for norovirus (NoV) from the summer and fall-winter season, respectively. The identified genotypes of NoV include GI-1, 4, 8, 9 and GII-4, 10, 11 (or 17), 13, 15 (or 16). None of fecal indicators was significantly correlated with NoV in groundwater. Among the tested fecal indicators, somatic coliphage (95.3%) showed an excellent true-negative rate of NoV occurrence. The combination of chemical, microbial and viral indicators increased the positive predictive value (50-100%). This study demonstrated a high prevalence of NoV in groundwater in metropolitan Seoul areas and characterized the positive and negative predictive values of a fecal indicator for predicting NoV prevalence.     

Characterization of sewage solid-associated viruses and behavior in natural waters

The association of bacteriophages and animal viruses with solids has been demonstrated to have a protective effect, resulting in enhanced survival in natural waters and resistance to inactivation by chlorine. In this study, attempts were made to differentiate solid-associated viruses and freely suspended viruses in secondarily treated sewage by the retention of sewage solids on membrane filters treated with fetal calf serum to prevent adsorption of freely suspended virus. Solid-associated viruses collected on membrane filters were eluted with pH 11.5, 0.05 M glycine buffer. The percentage of the total coliphage and animal virus associated with solids in secondarily treated sewage discharges ranged from < 1.0 to 24% and 3 to 100%, respectively. The largest quantity of solid-associated coliphage was attached to particles greater than 8.0 μm and less than 0.65 μm in size. Tapwater, lake water and estuarine water were all capable of eluting solid-associated coliphages. Elution of coliphages in marine water appeared to be related to the salinity of the water. Coliphages eluted from sewage solids in seawater could readsorb to naturally occurring marine sediment.     

Characterization of Enterococcus faecalis-infecting phages (enterophages) as markers of human fecal pollution in recreational waters

Enterophages are a novel group of phages that specifically infect Enterococcus faecalis and have been recently isolated from environmental water samples. Although enterophages have not been conclusively linked to human fecal pollution, we are currently characterizing enterophages to propose them as viral indicators and possible surrogates of enteric viruses in recreational waters. Little is known about the morphological or genetic diversity which will have an impact on their potential as markers of human fecal contamination. In the present study we are determining if enterophages can be grouped by their ability to replicate at different temperatures, and if different groups are present in the feces of different animals. As one of the main objectives is to determine if these phages can be used as indicators of the presence of enteric viruses, the survival rate under different conditions was also determined as was their prevalence in sewage and a large watershed. Coliphages were used as a means of comparison in the prevalence and survival studies. Results indicated that the isolates are mainly DNA viruses. Their morphology as well as their ability to form viral plaques at different temperatures indicates that several groups of enterophages are present in the environment. Coliphage and enterophage concentrations throughout the watershed were lower than those of thermotolerant coliforms and enterococci. Enterophage concentrations were lower than coliphages at all sampling points. Enterophages showed diverse inactivation rates and T₉₀ values across different incubation temperatures in both fresh and marine waters and sand. Further molecular characterization of enterophages may allow us to develop probes for the real-time detection of these alternative indicators of human fecal pollution.     

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution.     

Monitoring source water for microbial contamination: evaluation of water quality measures

Watershed management programs often rely on monitoring for a large number of water quality parameters to define contaminant issues. While coliforms have traditionally been used to identify microbial contamination, these indicators cannot discriminate among potential contaminant sources. Microbial source tracking (MST) can provide the missing link that implicates the sources of contamination. The objective of this study was to use a weight-of-evidence approach (land use analysis using GIS, sanitary surveys, traditional water quality monitoring, and MST targets) to identify sources of pollution within a watershed that contains a raw drinking water source. For the study watersheds, statistical analyses demonstrated that one measure each of particulate matter (turbidity, particle counts), organic matter (total organic carbon, dissolved organic carbon, UV(254) absorbance), and indicator organisms (fecal coliforms, enterococci) were adequate for characterizing water quality. While these traditional parameters were useful for assessing overall water quality, they were not intended to differentiate between microbial sources at different locations. In contrast, the MST targets utilized (Rhodococcus coprophilus, sorbitol-fermenting Bifidobacteria, and male-specific coliphages) pinpointed specific sources of microbial pollution. However, these targets could not be used for routine monitoring due to a high percentage of non-detects.     

Evaluation of rapid methods and novel indicators for assessing microbiological beach water quality

A broad suite of new measurement methods and indicators based on molecular measurement technology have been developed to assess beach water quality, but they have generally been subjected to limited testing outside of the laboratory in which they were developed. Here we evaluated 29 assays targeting a variety of bacterial, viral, and chemical analytes by providing the method developers with twelve blind samples consisting of samples spiked with known concentration of sewage or gull guano and negative controls. Each method was evaluated with respect to its ability to detect the target organism, absence of signal in the negative controls and repeatability among replicates. Only six of the 30 methods detected their targets in at least 75% of the samples while consistently determining the absence of the target in the negative controls. Among quantitative methods, QPCR for Bacteroides thetaiotamicron and Enterococcus detected by Luminex reliably identified all but one sample containing human fecal material and produced no false positive results. Among non-quantitative methods, the Enterococcus esp gene, the Bacteroidales human specific marker and culture-based coliphage were the most reliable for identifying human fecal material. We also found that investigator-specific variations of methods targeting the same organism often produced different results.     

Comparison of bacterial indicator analysis methods in stormwater-affected coastal waters

Membrane filtration (MF) and multiple tube fermentation (MTF) have been used for decades to measure indicator bacteria levels in beach water samples, but new methods based on chromogenic substrate (CS) technology are becoming increasingly popular. Only a few studies have compared results among these methods and they have generally been based on samples collected from a limited number of sites during dry weather. In this study, samples were collected from 108 sites the day after a major rainstorm, and three indicator bacteria (total coliforms (TCs), fecal coliforms (FCs) or E. coli, and enterococci (EC)) were each measured using MF, MTF, and CS. Sampling sites were selected using a stratified random design, stratified by open sandy beach, rocky shoreline, and beach areas near urban runoff outlets. The CS results were found to be highly correlated with both MF and MTF for all three indicators regardless of whether the samples were taken along open shoreline or near a runoff outlet. While correlated, TC values were higher using the CS method, consistent with other studies that have demonstrated false positives with this method. FC values were 12% lower with CS, reflecting the specificity of the CS method for E. coli rather than for the entire FC group. No significant differences were observed for EC, although some differences were observed within specific laboratories. Differences for all of these indicators were small enough that, when assessed categorically, there was more than 90% agreement between CS methods and either MF or MTF methods as to whether State of California Beach Water Quality Standards were met or exceeded.