Bacteriophage Transport in Sandy Soil and Fractured Tuff

Bacteriophage transport was investigated in laboratory column experiments using sandy soil, a controlled field study in a sandy wash, and laboratory experiments using fractured rock. In the soil columns, the phage MS-2 exhibited significant dispersion and was excluded from 35 to 40% of the void volume but did not adsorb. Dispersion in the field was similiar to that observed in the laboratory. The phage f2 was largely excluded from the porous matrix of the two fractured-rock cores studied, coming through 1.2 and 2.0 times later than predicted on the basis of fracture flow alone. Because of matrix diffusion, nonsorbing solutes were retarded by over a factor of three relative to fracture flow. The time for a solute tracer to equilibrate with the porous matrix of 6.5-cm-diameter by 25-cm-long cores was measured in days. Results of both granular-medium and fractured-rock experiments illustrate the inability of a solute tracer to provide estimates for dispersion and effective porosity that are applicable to a colloid. Bacteriophage can be used to better estimate the maximum subsurface transport rate of colloidal contaminants through a porous formation.     

Estimation of bioaerosol risk of infection to residents adjacent to a land applied biosolids site using an empirically derived transport model

AIM: The purpose of this study was to develop an empirically derived transport model, which could be used to predict downwind concentrations of viruses and bacteria during land application of liquid biosolids and subsequently assess microbial risk associated with this practice. METHODS AND RESULTS: To develop the model, coliphage MS-2 and Escherichia coli were aerosolized after addition to water within a biosolids spray application truck, and bioaerosols were collected at discrete downwind distances ranging from 2 to 70 m. Although coliphage were routinely detected, E. coli did not frequently survive aerosolization. Data on aerosolized coliphage was then used to generate a virus transport model. Risks of infection were calculated for various ranges of human virus concentrations that could be found in biosolids. CONCLUSIONS: A conservative estimate at 30.5 m (assumed to be nearest adjacent residences) downwind, resulted in risks of infection of 1 : 100,000, to the more realistic 1 : 10,000,000 per exposure. Conservative annual risks were calculated to be no more than 7 : 100,000 where as a more realistic risk was no greater than 7 : 10,000,000. Overall, the viral risk to residences adjacent to land application sites appears to be low, both for one time and annual probabilities of infection. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a simple approach towards modelling viral pathogens aerosolized from land applied liquid biosolids, and offers insight into the associated viral risk.     

Inactivation of feline calicivirus and adenovirus type 40 by UV radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm(2), respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm(2) in treated groundwater. A dose of 103 mJ/cm(2) was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

Inactivation of Feline Calicivirus and Adenovirus Type 40 by UV Radiation

Little information regarding the effectiveness of UV radiation on the inactivation of caliciviruses and enteric adenoviruses is available. Analysis of human calicivirus resistance to disinfectants is hampered by the lack of animal or cell culture methods that can determine the viruses' infectivity. The inactivation kinetics of enteric adenovirus type 40 (AD40), coliphage MS-2, and feline calicivirus (FCV), closely related to the human caliciviruses based on nucleic acid organization and capsid architecture, were determined after exposure to low-pressure UV radiation in buffered demand-free (BDF) water at room temperature. In addition, UV disinfection experiments were also carried out in treated groundwater with FCV and AD40. AD40 was more resistant than either FCV or coliphage MS-2 in both BDF water and groundwater. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in BDF water were 109, 55, and 16 mJ/cm², respectively. The doses of UV required to achieve 99% inactivation of AD40, coliphage MS-2, and FCV in groundwater were slightly lower than those in BDF water. FCV was inactivated by 99% by 13 mJ/cm² in treated groundwater. A dose of 103 mJ/cm² was required for 99% inactivation of AD40 in treated groundwater. The results of this study indicate that if FCV is an adequate surrogate for human caliciviruses, then their inactivation by UV radiation is similar to those of other single-stranded RNA enteric viruses, such as poliovirus. In addition, AD40 appears to be more resistant to UV disinfection than previously reported.     

Male-specific coliphages as indicators of thermal inactivation of pathogens in biosolids

Male-specific (F+) coliphages have been proposed as a candidate indicator of fecal contamination and of virus reduction in waste treatment. However, in this and earlier work with a laboratory thermophilic anaerobic digester, a heat-resistant fraction of F+ coliphage populations indigenous to municipal wastewater and sludge was evident. We therefore isolated coliphages from municipal wastewater sludge and from biosolid samples after thermophilic anaerobic digestion to evaluate the susceptibility of specific groups to thermal inactivation. Similar numbers of F+ DNA and F+ RNA coliphages were found in untreated sludge, but the majority of isolates in digested biosolids were group I F+ RNA phages. Separate experiments on individual isolates at 53 degrees C confirmed the apparent heat resistance of group I F+ RNA coliphages as well as the susceptibility of group III F+ RNA coliphages. Although few F+ DNA coliphages were recovered from the treated biosolid samples, thermal inactivation experiments indicated heat resistance similar to that of group I F+ RNA phages. Hence, F+ DNA coliphage reductions during thermophilic anaerobic digestion are probably related to mechanisms other than thermal inactivation. Further studies should focus on the group III F+ RNA coliphages as potential indicators of reductions of heat-resistant pathogens in thermal processes for sludge treatment.     

Characteristics and biogas production potential of municipal solid wastes pretreated with a rotary drum reactor

This study was conducted to determine the characteristics and biogas production potential of organic materials separated from municipal solid wastes using a rotary drum reactor (RDR) process. Four different types of wastes were first pretreated with a commercial RDR system at different retention times (1, 2 and 3 d) and the organic fractions were tested with batch anaerobic digesters with 2.6 g VS L(-1) initial loading. The four types of waste were: municipal solid waste (MSW), a mixture of MSW and paper waste, a mixture of MSW and biosolids, and a mixture of paper and biosolids. After 20 d of thermophilic digestion (50+/-1 degrees C), it was found that the biogas yields of the above materials were in the range of 457-557 mL g VS(-1) and the biogas contained 57.3-60.6% methane. The total solid and volatile solid reductions ranged from 50.2% to 65.0% and 51.8% to 66.8%, respectively. For each material, the change of retention time in the RDR from 1 to 3d did not show significant (alpha=0.05) influence on the biogas yields of the recovered organic materials. Further studies are needed to determine the minimum retention time requirements in the RDR system to achieve effective separation of organic from inorganic materials and produce suitable feedstock for anaerobic digesters.     

Evaluation of biotracers to monitor effluent retention time in constructed wetlands

AIMS: With concern surrounding the environmental impact of chemical tracers on the aquatic environment, this paper presents the initial evaluation of biotracers used to determine the effluent retention time, an important performance indicator, in a Free Water Surface Constructed Wetland. METHODS AND RESULTS: Production of the biotracers, coliphage MS2, and the bacteriophage of Enterobacter cloacae and antibiotic resistant endospores of Bacillus globigii is described in detail. Their subsequent use in three separate tracer experiments - January, March and June (2000) - revealed the variability of retention time with respect to effluent flow. The biotracer MS2 showed the constructed wetland had a retention time of 8-9 h at a mean discharge of 0.9 l s-1, increasing to 10-12 h at a mean discharge 0.3 l s-1. A similar retention of 9-10 h at a mean discharge of 0.3 l s-1 was calculated for the Ent. cloacae phage. In contrast, use of endospores revealed considerably longer retention times at these mean discharge rates; 12-24 h and 36-48 h, respectively. CONCLUSION: Biotracers could provide a useful and environmentally friendly technique to monitor effluent retention in constructed wetlands. At this stage the phage tracers appear particularly promising due to ease of isolation and recovery. SIGNIFICANCE AND IMPACT OF THE STUDY: Initial results are encouraging and have highlighted the potential of biotracers as alternatives to chemical tracers, even in microbially-rich waters.     

Effect of plant growth on dehydration rates and microbial populations in sewage biosolids

The high water content of sewage biosolids makes them expensive to transport. Two experiments were done to see if it was practical to use transpiration by plants as a low cost method to dehydrate biosolids. (i) To assess biosolids as a growth-medium for plants, maize, beans, pumpkin, forage oats and an annual ryegrass were grown in pots of fresh biosolids. Plant growth varied between species and potassium deficiency was found to be a limiting factor for barley. Roots were slow to penetrate anoxic biosolids in the bottom of the pots. (ii) Dehydration rates were measured in 30 litre boxes of biosolids from two different sources. Boxes were planted with maize or beans, or kept fallow. Despite the high (73-83%) initial water content of the biosolids, plants were susceptible to wilting on warm days which suggested that a significant proportion of the water in biosolids is not freely available to plants. Shrinkage caused a major reduction in biosolids volume in both experiments. When change in volume of the biosolids residue was accounted for, there was a 56% average water loss in planted treatments and 34% water loss in the fallow treatment. This indicates that planting may have some potential as a technique to accelerate dehydration of biosolids. Water contents were not reduced sufficiently to influence biosolids microbial populations.     

Leaching of Cryptosporidium parvum oocysts, Escherichia coli, and a Salmonella enterica serovar Typhimurium bacteriophage through intact soil cores following surface application and injection of slurry

Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions.     

A national study on the residential impact of biological aerosols from the land application of biosolids

AIMS: The purpose of this study was to evaluate the community risk of infection from bioaerosols to residents living near biosolids land application sites. METHODS AND RESULTS: Approximately 350 aerosol samples from 10 sites located throughout the USA were collected via the use of six SKC Biosamplers. Downwind aerosol samples from biosolids loading, unloading, land application and background operations were collected from all sites. All samples were analysed for the presence of HPC bacteria, total coliform bacteria, Escherichia coli, Clostridium perfringens, coliphage, enteroviruses, hepatitis A virus and norovirus. Total coliforms, E. coli, C. perfringens and coliphage were not detected with great frequency from any sites, however, biosolids loading operations resulted in the largest concentrations of these aerosolized microbial indicators. Microbial risk analyses were conducted on loading and land application operations and their subsequent residential exposures determined. CONCLUSIONS: The greatest annual risks of infection occurred during loading operations, and resulted in a 4 x 10(-4) chance of infection from inhalation of coxsackievirus A21. Land application of biosolids resulted in risks that were <2 x 10(-4) from inhalation of coxsackievirus A21. Overall bioaerosol exposure from biosolids operations poses little community risk based on this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluated the overall incidence of aerosolized micro-organisms from the land application of biosolids and subsequently determined that microbial risks of infection were low for residents close to biosolids application sites.     

A new approach to model the spatiotemporal development of biofilm phase in porous media

Bacteria can exist within biofilms that are attached to the solid matrix of a porous medium. Under certain conditions, the biomass can fully occupy the pore space leading to reduced hydraulic conductivity and mass transport. Here, by treating biofilm as a growing, high-viscosity phase, a novel macroscopic approach to model biofilm spatial expansion and its corresponding effects on porous medium hydraulic properties is presented. The separate yet coupled flow of the water and biofilm phases is handled by using relative permeability curves that allow for biofilm movement within the porous medium and bioclogging effects. Fluid flow is governed by Darcy's law and component transport is set by the convection-diffusion equation reaction terms for each component. Here, the system of governing equations is solved by using a commercial multiphase flow reservoir simulator, which is used to validate the model against published laboratory experiments. A comparison of the model and experimental observations reveal that the model provides a reasonable means to predict biomass development in the porous medium. The results reveal that coupled flow of water and movement of biofilm, as described by relative permeability curves, is complex and has a large impact on the development of biomass and consequent bioclogging in the porous medium.     

Comparison between pine bark and coconut husk sorption capacity of metals and nitrate when mixed with sewage sludge

Waste products such as biosolids and wood wastes have been frequently used in container production of plants. The use of biosolids in agriculture is a beneficial mean of recycling the by-products of waste-water treatment. However, care must be taken to avoid environmental or human health problems via run-off and leaching. The objective of this work is to compare the retention capacity of cadmium, lead, zinc and nitrate between pine bark (PB) and coconut fibre (F) when mixed with increasing amounts of composted sewage sludge (CSS) (0%, 15% and 30% (v/v)). Substrates were packed into leaching columns and irrigated with deionised water every 2 days. Leachates were collected during 1 month, and nitrate, Zn, Cd, Pb, EC and pH were monitored along the experiment. PB columns leached lower amount of nitrate than the coconut fibre ones. The same trend was observed for Zn, Cd and Pb. It could be said, that in order to minimize the environmental risks of using sewage sludges our results indicate that it is preferred to mix the sludge with pine bark instead than with coconut husk.     

Inactivation of coliphage MS-2 and poliovirus by copper, silver, and chlorine.

The efficacy of electrolytically generated copper and silver ions (400 and 40 micrograms/L, respectively) was evaluated separately and in combination with free chlorine (0.2 and 0.3 mg/L) for the inactivation of coliphage MS-2 and poliovirus type 1 in water at pH 7.3. The inactivation rate was calculated as log10 reduction/min: k = -(log10 Ct/C0)/t. The inactivation of both viruses was at least 100 times slower in water containing 400 and 40 micrograms/L copper and silver, respectively (k = 0.023 and 0.0006 for MS-2 and poliovirus, respectively), compared with water containing 0.3 mg/L free chlorine (k = 4.88 and 0.036). Significant increases in the inactivation rates of both viruses were observed in test systems containing 400 and 40 micrograms/L copper and silver, respectively, with 0.3 mg/L free chlorine when compared with the water systems containing either metals or free chlorine alone. Poliovirus was approximately 10 times more resistant to the disinfectants than coliphage MS-2. This observation suggests either a synergistic or an additive effect between the metals and chlorine for inactivation of enteric viruses. Use of copper and silver ions in water systems currently used in swimming pools and spas may provide an alternative to high levels of chlorination.     

A note on gentamicin susceptibility testing: the validity of the MS-2 system and Rosco Neosensitabs agar diffusion

The MS-2 automated system was compared with Rosco Neosensitabs agar diffusion for the gentamicin susceptibility of a group of 267 organisms isolated from clinical material. With Rosco Neosensitabs agar diffusion agreement with reference minimal inhibitory concentrations was 98.5%, while with the MS-2 system it was 100% if calcium and magnesium-supplemented MS-2 broth was used.     

Virus persistence in groundwater

More than 50% of the outbreaks of waterborne disease in the United States are due to the consumption of contaminated groundwater. An estimated 65% of the cases in these outbreaks are caused by enteric viruses. Little, however, is known about the persistence of viruses in groundwater. The purpose of this study was to determine whether measurable chemical and physical factors correlate with virus survival in groundwater. Groundwater samples were obtained from 11 sites throughout the United States. Water temperature was measured at the time of collection. Several physical and chemical characteristics, including pH, nitrates, turbidity, and hardness, were determined for each sample. Separate water samples were inoculated with each of three viruses (poliovirus 1, echovirus 1, and MS-2 coliphage) and incubated at the in situ groundwater temperature; selected samples were also incubated at other temperatures. Assays were performed at predetermined intervals over a 30-day period to determine the number of infective viruses remaining. Multiple regression analysis revealed that temperature was the only variable significantly correlated with the decay rates of all three viruses. No significant differences were found among the decay rates of the three viruses, an indication that MS-2 coliphage might be used as a model of animal virus survival in groundwater.     

A note on gentamicin susceptibility testing: the validity of the MS-2 system and Rosco Neosensitabs agar diffusion

The MS-2 automated system was compared with Rosco Neosensitabs agar diffusion for the gentamicin susceptibility of a group of 267 organisms isolated from clinical material. With Rosco Neosensitabs agar diffusion agreement with reference minimal inhibitory concentrations was 98.5%, while with the MS-2 system it was 100% if calcium and magnesium-supplemented MS-2 broth was used.     

Virus persistence in groundwater.

More than 50% of the outbreaks of waterborne disease in the United States are due to the consumption of contaminated groundwater. An estimated 65% of the cases in these outbreaks are caused by enteric viruses. Little, however, is known about the persistence of viruses in groundwater. The purpose of this study was to determine whether measurable chemical and physical factors correlate with virus survival in groundwater. Groundwater samples were obtained from 11 sites throughout the United States. Water temperature was measured at the time of collection. Several physical and chemical characteristics, including pH, nitrates, turbidity, and hardness, were determined for each sample. Separate water samples were inoculated with each of three viruses (poliovirus 1, echovirus 1, and MS-2 coliphage) and incubated at the in situ groundwater temperature; selected samples were also incubated at other temperatures. Assays were performed at predetermined intervals over a 30-day period to determine the number of infective viruses remaining. Multiple regression analysis revealed that temperature was the only variable significantly correlated with the decay rates of all three viruses. No significant differences were found among the decay rates of the three viruses, an indication that MS-2 coliphage might be used as a model of animal virus survival in groundwater.     

Transport of bacteria in porous media and its enhancement by surfactants for bioaugmentation: A review

The success of bioaugmentation processes for groundwater bioremediation requires efficient transport of bacteria in the subsurface environment. In this paper, the factors that influence transport of bacterial cells in porous media are reviewed and the effects of surfactants on the transport are discussed. Movement of bacterial cells in porous media is a process driven by advection and hydrodynamic dispersion forces of fluids. Immobilization of bacterial cells takes place due to processes such as adsorption and straining. Blocking and ripening along with bacterial migration process decrease and increase the retention of cells in porous media, respectively. Physicochemical properties of the porous media, groundwater chemistry, and properties of the bacterial cells affect the transport behavior. Surfactants have the potential to modify bacterial surface properties for both bacterial cells and medium solids, and thus enhance bacterial transport.     

Evaluation of the Edgegard Laminar Flow Hood

In a horizontal back-to-front flow high-efficiency particulate air-filtered laminar hood, it is shown that a Blake bottle obstruction to the air flow causes a downstream cone of turbulent air which can draw microbial contamination into the work area of the hood. In controlled experiments, contamination with T3 coliphage was reduced by a series of perforations around the open edge of the hood which eliminates the cone of turbulent air. The average reduction in phage counts was 90.75, 86.79, 91.12, and 98.92%, depending upon the site of nebulization. The phage counts were reduced in 48 of the 51 tests.     

Pretreatment to reduce somatic salmonella phage interference with FRNA coliphage assays: successful use in a one-year survey of vulnerable groundwaters

Somatic salmonella (SS) phages were commonly found in higher numbers than F-specific RNA (FRNA) coliphages in a multi-site survey of contamination-vulnerable groundwaters. The relative abundance of SS phages required that a pretreatment procedure be implemented to reduce the SS phage content of samples before FRNA coliphage assay with Salmonella typhimurium WG49. Pretreatment involved selective SS phage removal by Salm. typhimurium WG45 cells. This pretreatment proved effective in producing interference-free samples throughout the one-year survey period and in seeded evaluation, was shown not to affect the detection of representative FRNA coliphage MS2. During the survey, 30 groundwater sites located in the continental United States, Puerto Rico and the Virgin Islands were examined for FRNA coliphages and SS phages at monthly intervals. FRNA coliphages were detected at six of the 30 sites and in 33 of 329 monthly samples. SS phages were also detected at six sites and in 28 of 329 monthly samples. Five of the phage-positive sites were positive for both phage groups. At those five sites, 58 monthly samples were collected during the survey period. Those 58 samples yielded an average FRNA coliphage concentration of 140 pfu per 100 1 of groundwater as compared to an average SS phage concentration of 565 pfu per 100 1 of groundwater. Twenty of the 58 samples were positive for both FRNA coliphages and SS phages. In those samples, FRNA coliphages were more abundant in five samples; SS phages were more abundant in 15 samples. Because these results demonstrate that SS phage levels may often exceed FRNA coliphage levels in environmental waters, it is clear that SS phage removal procedures will greatly enhance the effectiveness of the WG49-based FRNA coliphage assay.