Demonstration of solids-associated virus in wastewater and sludge.

Data presented demonstrate the relatively high multiplicity of solids-associated virus in field samples, i.e., wastewater, sludge, and soils. Influent, effluent, and chlorinated effluent samples showed 16.1 to 100% of the total virus demonstrated in samples to be solids associated. Three techniques for freeing solids-associated virus are described and compared. Using sonication of solids and polyethylene glycol concentration, virus was demonstrated in fully digested sludge (60 days at 34 C), sand at the site of a sewer leak, and dried sludge cake and mud 900 m downstream from a sewage disposal site. These data emphasize the inadequacy of virus concentration techniques that do not include the processing of solids. In situ elution failed to free solids-associated virus.     

Distribution of viruses associated with particles in waste water.

The distribution of solids-associated viruses in wastewater was studied to determine the effect of treatment processes on viruses associated with solids. Solids less than 0.3 micrometers in diameter were separated from the liquid phase of each sample by using a continuous-flow centrifuge. The percentage of virus associated with solids larger than 0.3 micrometers decreased from 28% in the influent to 3.4% in unchlorinated effluent, and this was accompanied by a 92% decrease in the total concentration of virus. These results indicate that the original solids-associated virus as well as that is secondarily adsorbed to mixed liquor-suspended solids is lost during clarification. The total concentration of virus was reduced by 82% by chlorination, and the percentage of virus associated with solids increased to 7.7% upon chlorination, indicating some protection due to association with particles larger than 0.3 micrometers. When a suspension of fecal particles and a 0.22-micrometers filtrate of a fecal homogenate were sonicated, a threefold increase in virus titer was observed in each. This demonstrated that viruses may be attached to particles smaller than 0.22 micrometers. Thus, small viral aggregates or viruses attached to submicron particles represented the major portion of solids-associated virus in treated sewage.     

Effect of sludge type on poliovirus association with and recovery from sludge solids

Sludge type was found to affect the degree of association between seeded poliovirus type 1 (LSc) and sludge solids. The mean percent of solids-associated viruses for activated sludge mixed liquors, anaerobically digested sludges, and aerobically digested percent of solids-associated viruses for activated sludge mixed liquors, anaerobically digested sludges, and aerobically digested sludges was 57.2, 70.4, and 94.7, respectively. The degree of association between poliovirus and sludge solids was significantly greater for aerobically digested sludges than for the other two sludge types. Sludge solids associated viruses were eluted using 0.05 M glycine buffer, pH 10.5-11.0, and subsequently concentrated by organic flocculation. The effectiveness of the glycine method in the recovery of solids-associated viruses was also found to be affected by sludge type. Significantly lower mean poliovirus recovery was found for aerobically digested sludges (14.5%) than for mixed liquors or anaerobically digested sludges (72.3 and 60.2%, respectively). The eluent used in the method was not as effective in dissociating the virus from aerobic sludge solids as it was for the other two sludge types. All other virus adsorption-elution steps of the method (i.e., virus concentration steps) were equally effective in poliovirus recovery for all three sludge types. It is suggested that future methods developed for the recovery of viruses from sludges be evaluated for the various sludge types likely to be tested.     

Viruses in sewage: effect of phosphate removal with calcium hydroxide (lime).

During calcium hydroxide (lime) treatment (pH 9.6 to 10.5) of wastewaters for phosphate removal there was also a two-log removal of added poliovirus (type I, Sabin) from effluents. A similar virus reduction was seen in the sludge generated in these experiments. However, in view of the limitations of techniques for virus recovery from sludge, only a small portion of the infectious virus present in lime sludge may have been detected. Storage of lime sludge at 28 degrees C for up to 48 h produced no appreciable reduction in the virus titre. Five sets of field samples of sewage, effluents, and sludge from a sewage treatment plant (Kemptville, Ont.) which utilizes lime for phosphate removal were also examined for indigenous viruses being BS-C-1 cells. All of the sample of lime sludge and 80% of the samples of both sewage and lime-treated effluent revealed virus; after chlorination only 20% of the lime-treated effluent samples were positive for virus. In contrast, in an earlier study with essentially the same experimental set up, 76% of the sample of chlorinated primary effluent were found to contain virus. Because of the easily detectable quantities of infectious virus in lime sludge and due to the lack of virus inactivation during storage of such sludge, caution must be exercised in its handling and disposal.     

Association of Enteroviruses with Natural and Artificially Introduced Colloidal Solids in Water and Infectivity of Solids-Associated Virions

Encephalomyocarditis viruses adsorb to introducted organic and inorganic solids in water over a wide range of pH and with various concentrations and species of metal cations. Visible flocculation of solids was not a prerequisite for significant virus association. Virus adsorption to natural solids in various types of natural waters was significant but variable. Clay-adsorbed virus retained its infectivity in tissue culture monolayers. These solids-associated viruses also retained infectivity in mice.     

Inactivation of poliovirus and other enteroviruses by solutions of sodium fluoride at low pH

Solutions of sodium fluoride at pH 3 to 4 inactivated enteroviruses, whereas other sodium salts had little or no effect on virus infectivity. Solutions of potassium fluoride also inactivated viruses under similar conditions. Light, temperature, and the presence of organic compounds such as detergents and fecal matter did not affect inactivation of virus by 0.4 M solutions of sodium fluoride at pH 3. to 4. Decreasing the sodium fluoride concentration below 0.04 M or raising the pH above 4 reduced the viricidal properties of the solutions. Virus adsorbed to membrane filters and sludge flocs could not be recovered after treatment of solids-associated virus with solutions of sodium fluoride.     

Inactivation of poliovirus and other enteroviruses by solutions of sodium fluoride at low pH.

Solutions of sodium fluoride at pH 3 to 4 inactivated enteroviruses, whereas other sodium salts had little or no effect on virus infectivity. Solutions of potassium fluoride also inactivated viruses under similar conditions. Light, temperature, and the presence of organic compounds such as detergents and fecal matter did not affect inactivation of virus by 0.4 M solutions of sodium fluoride at pH 3. to 4. Decreasing the sodium fluoride concentration below 0.04 M or raising the pH above 4 reduced the viricidal properties of the solutions. Virus adsorbed to membrane filters and sludge flocs could not be recovered after treatment of solids-associated virus with solutions of sodium fluoride.     

On-line fluorescence profile of aerobic sludge digestion

An online fluorometer designed for following intracellular NAD(P)H was used to monitor aerobic sludge digestion experiments. The fluorescence showed an initial rise to a high plateau, a sharp decline after staying at the plateau for 20-60 h, and a trailing very slow decrease. The characteristic fluorescence profile was shown to result mainly from the solids-associated fluorescence, after ruling out other factors such as pH, temperature, and supernatant fluorescence. The fluorescence profile was, however, not a mere result of the decreasing solids concentration. The varying sludge viability and population composition (e.g., the decay of heterotrophs and the increasing fraction of nitrifiers) played important roles. The fluorescence profile correlated well with the profile of the viable heterotrophic cell number concentration evaluated with TSB-agar plates. The initial increase of the number concentration was attributed to the growth of multiple small bacteria from the lysate of each large microorganism, which was demonstrated in the experiments with baker's yeast as the starting culture for digestion. The fluorescence profiles observed in the yeast experiments were similar to those in the sludge experiments. Responding to glucose additions and the switch from aerobic to anaerobic conditions, the yeast systems showed typical step increases of fluorescence as expected from the change of NAD(P)H level associated with heterotrophic metabolism. However, no such fluorescence responses were detectable in the sludge digestion systems. NAD(P)H were thus uncertain to be responsible for the online fluorescence observed. Nonetheless, the initial fluorescence plateau corresponded to the period of rapid digestion and, for the plant studied, the EPA regulation criteria of VSS reduction >38% and/or SOUR <1.5 mg of O(2) (g of TS)(-)(1) h(-)(1) were satisfied at the end of the plateau. The online fluorescence provides an effective means of monitoring the aerobic sludge digestion process.     

Characterization and biological treatment of ceramic industry wastewater

Ceramic industry wastewaters not only contain high suspended and total solids but also significant amounts of dissolved organics resulting in high BOD or COD loads. Suspended solids can be removed from the wastewater by chemical precipitation. However, dissolved BOD/COD compounds can only be removed by biological or chemical oxidation. Effluent wastewater from chemical sedimentation stage of EGE CERAMIC industry was characterized and subjected to biological treatment in a laboratory scale activated sludge unit. Experiments were conducted at different hydraulic and solids retention times. The best results were obtained with Š_c=20 h of hydraulic and Š_c=20 days of solids retention times (sludge age) resulting in effluent COD concentration of 40 mg/l from a feed wastewater of 720 mg/l COD content. The suspended solids content of the activated sludge effluent was approximately 52 mg/l.     

Application of a bioluminescence ATP assay in brewery wastewater treatment studies

The importance of having a rapid method for determining the viable biomass in activated-sludge wastewater treatment plants (WWTP) for process control and development is well recognized. The firefly bioluminescence ATP assay has been proposed for this purpose. Such an assay using partially purified firefly luciferase and synthetic firefly luciferin for the bioluminescence reaction, a liquid scintillation counter in the out-of-coincidence mode as luminescence detector, and a sludge ATP extraction technique involving dimethyl sulfoxide at room temperature is described. Experiments with several pure bacteria cultures were done to demonstrate the feasibility of applying this assay to activated sludge to activated sludge WWTP investigation and control. The ATP content of samples taken from various points in a 350000 gal/day brewery activated-sludge WWTP was monitored for 4.5 months. Good linear correlation between ATP and mixed-liquor suspended solids, return sludge suspended solids, and effluent suspended solids were observed. Percentage viabilities of the various sludge samples were derived from the ATP results.     

Assessment of recovery efficiency of beef extract reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation procedure

This study was designed to assess the capacity of beef extract reagents to form flocs suitable for virus adsorption. Reagent comparisons resulted in the establishment of a modified organic flocculation procedure to concentrate viruses desorbed from sewage sludge solids with currently available modified powdered beef extracts. The method, based on supplementation with paste beef extract floc, achieved virus recoveries comparable to those obtained with powdered beef extract produced before a 1979 change in the manufacturing process. When primary settled sludge solids originating from mostly domestic waste were eluted with an unsupplemented modified powdered beef extract, high virus recovery efficiency was observed upon concentration by organic flocculation. This appreciable increase might have been due to floc-forming substances that were present in the primary settled sludge. These substances did not appear to be present in settled sludge collected from biologically treated wastes. Apparently, the floc-forming substances had been either removed or substantially altered during biological treatment.     

Assessment of recovery efficiency of beef extract reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation procedure.

This study was designed to assess the capacity of beef extract reagents to form flocs suitable for virus adsorption. Reagent comparisons resulted in the establishment of a modified organic flocculation procedure to concentrate viruses desorbed from sewage sludge solids with currently available modified powdered beef extracts. The method, based on supplementation with paste beef extract floc, achieved virus recoveries comparable to those obtained with powdered beef extract produced before a 1979 change in the manufacturing process. When primary settled sludge solids originating from mostly domestic waste were eluted with an unsupplemented modified powdered beef extract, high virus recovery efficiency was observed upon concentration by organic flocculation. This appreciable increase might have been due to floc-forming substances that were present in the primary settled sludge. These substances did not appear to be present in settled sludge collected from biologically treated wastes. Apparently, the floc-forming substances had been either removed or substantially altered during biological treatment.     

Suitability of aquaculture effluent solids mixed with cardboard as a feedstock for vermicomposting

Recirculating aquaculture systems are highly intensive culture systems that actively filter and reuse water, thus minimizing water requirements and creating relatively small volumes of concentrated waste (compared to flow-through aquaculture systems). Vermicomposting, which uses earthworms to stabilize and transform organic wastes into valuable end-products, has been proposed as an alternative treatment technology for high-moisture-content organic wastes from agricultural, industrial and municipal sources. This study was conducted to determine if the effluent solids from a large recirculating aquaculture facility (Blue Ridge Aquaculture, Martinsville, Virginia) were suitable for vermicomposting using the earthworm Eisenia fetida. In two separate experiments, worms were fed mixtures of solids removed from aquaculture effluent (sludge) and shredded. Mixtures containing 0%, 5%, 10%, 15%, 20%, 25%, and 50% aquaculture sludge (dry weight basis) were fed to the worms over a 12-week period and their growth (biomass) was measured. Worm mortality, which occurred only in the first experiment, was not influenced by feedstock sludge concentration. In both experiments worm growth rates tended to increase with increasing sludge concentration, with the highest growth rate occurring with feedstocks containing 50% aquaculture sludge. Effluent solids from recirculating aquaculture systems mixed with shredded cardboard appear to be suitable feedstocks for vermicomposting.     

Simple apparatus for collecting estuarine sediments and suspended solids to detect solids-associated virus

Laboratory trials of a new sampler for collection of estuarine sediment-associated virus resulted in a recovery effectiveness averaging 30% for two enteroviruses and rotavirus SA11. A minimal recovery potential of 54% was calculated when losses caused by virus concentration procedure inadequacies were excluded. Both sediment-associated and suspended solids-associated viruses were collected with the sampler. Recoveries of 61 and 60% poliovirus and rotavirus, respectively, were obtained from salt water-suspended, solids-associated virus. The unique advantage of the sampler for selective collection of virus-associated top layers of sediment, plus collection over extensive areas, resulted in recovery of more virus than was obtained with a commonly used dredge-type sampler.     

The application of constant recycle solids concentration in activated sludge process.

The applicability of the model derived by Ramanathan and Gaudy (Biotechnol. Bioeng., 11, 207, (1969)) for completely mixed activated sludge treatment holding the recycle solids concentration as a system constant was investigated using an actual industrial organic wastewater. Short-term experiments were conducted at various dilution rates (1/8, 1/6, 1/4, 1/2, 1/1.5 hr-1) for two recycle solids concentration values (5000 and 7000 mg/liter). The influent substrate concentration was maintained at 1000 mg/liter COD and the hydraulic recycle ratio- alpha, was kept at 0.3. It was found that for bottling plant (Pepsi Cola) wastewaters, a steady state with respect to reactor biological solids and effluent COD, at different dilution rates, could be attained, lending experimental evidence to the assumption that a steady state could be reached in developing the model and also affecting the applicability of the model in industrial organic wastewater. The reactor biological solids and effluent COD calculated from the model closely agreed with the observed values at dilution rates lower than 0.5 hr-1. Operation at dilution rates higher than 0.5 hr-1 will washout the biological solids from the reactor and the recycle substrate concentration will be apparent if the concentration of XR were not increased.     

A multiple extraction--centrifugation method for the recovery of viruses from waste water treatment plant effluents and sludges

Absorption of 14C-labelled poliovirus-2 to sedimentable solids of primary sludge samples collected from a secondary treatment facility during a 6-month period averaged 94%; for anaerobically digested sludge, 99%. The extent of virus adsorption was influenced by the amount of solids. Maximal adsorption occurred at or above 0.5% solids with sludge diluted with deionized water and above 1.5% solids when diluted with the respective particle-free sludge supernatants. A Tris-HCl buffer containing NaCl, glycerol, and serum was found to efficiently elute poliovirus-2 from primary sludge solids. By means of re-extraction and concentration by centrifugation (the TEC procedure), the average recoveries of poliovirus-2 were 92-94% based upon either infectivity or radioactivity analyses. Similarly, recoveries were 90-92% for poliovirus-2 in digested sludge. Maximum elution was dependent upon all four TEC buffer components and the restriction of solids to less than or equal to 1.0%. The procedure was found to be more efficient than glycine-NaOH and Freon procedures or elution with beef embryo extract. As adapted for effluents the procedure increased the yield and improved the consistency of virus recovery. The arithmetic mean titers and obtained during a monitoring study for primary and digested sludge were 4.2 X 10(5) and 5.1 X 10(3) plaque-forming units (pfu)/L; for primary, secondary, and final effluents 2.3 X 10(5), 4.7 X 10(3), and 4.7 X 10(2) pfu/L, respectively.     

Elimination of human enteric viruses during conventional waste water treatment by activated sludge

The present study was undertaken to determine if viruses were selectively eliminated during waste water treatment. Human enteric viruses were detected at all steps of treatment in a conventional activated sludge waste water treatment plant. Liquid overlays and large volume sampling with multiple passages on BGM cells permitted the detection of poliovirus (serotypes 1, 2, and 3), coxsackievirus B (serotypes 1, 2, 3, 4, and 5), and echovirus (serotypes 3, 14, and 22), as well as reoviruses. The mean virus concentration was 95.1 most probable number of infectious units per litre (mpniu/L) in raw sewage, 23.3 in settled water, 1.4 in effluent after activated sludge treatment, and 40.3 mpniu/L in sludge samples. All samples of raw sewage and settled water, 79% of effluent water, and 94% of sludge samples contained viruses. The mean reduction was 75% after settling and 98% after activated sludge treatment. Poliovirus type 3 was rarely isolated after the activated sludge treatment, but was still detected in about one-third of the sludge samples. Reoviruses and coxsackieviruses were detected at similar rates from all samples and appear to be more resistant to the activated sludge treatment than poliovirus type 3. Poliovirus types 1 and 2 were present in almost every sample of raw sewage and settled water and still found in about half of the effluent and sludge samples, indicating a level of resistance similar to that of reoviruses and coxsackieviruses.     

Simple apparatus for collecting estuarine sediments and suspended solids to detect solids-associated virus.

Laboratory trials of a new sampler for collection of estuarine sediment-associated virus resulted in a recovery effectiveness averaging 30% for two enteroviruses and rotavirus SA11. A minimal recovery potential of 54% was calculated when losses caused by virus concentration procedure inadequacies were excluded. Both sediment-associated and suspended solids-associated viruses were collected with the sampler. Recoveries of 61 and 60% poliovirus and rotavirus, respectively, were obtained from salt water-suspended, solids-associated virus. The unique advantage of the sampler for selective collection of virus-associated top layers of sediment, plus collection over extensive areas, resulted in recovery of more virus than was obtained with a commonly used dredge-type sampler.     

Development of quantitative methods for the detection of enteroviruses in sewage sludges during activation and following land disposal.

The development and evaluation of methods for the quantitative recovery of enteroviruses from sewage sludge are reported. Activated sewage sludge solids were collected by centrifugation, and elution of the solid-associated virus was accomplished by mechanical agitation in glycine buffer at pH 11.0. Eluted viruses were concentrated either onto an aluminum hydroxide floc or by association with a floc which formed de novo upon adjustment of the glycine eluate to pH 3.5. Viruses which remained in the liquid phase after lowering the pH of glycine eluate were concentrated by adsorption to and elution from membrane filters. The method of choice included high pH glycine elution and subsequent low pH concentration; it yielded an efficiency of recovery from activated sludge of 80% for poliovirus type 1, 68% for echovirus type 7, and 75% for coxsackievirus B3. This method was used to study the survival of naturally occurring virus in sludge at a sewage treatment plant and after subsequent land disposal of the solids after aerobic digestion. Reduction of enterovirus titers per gram (dry weight) of solids were modest during sludge activation but increased to a rate of 2 log 10/week after land disposal.