Recovery of indigenous enteroviruses from raw and digested sewage sludges

We examined different types of raw sewage sludge treatment, including consolidation, anaerobic mesophilic digestion with subsequent consolidation, and aerobic-thermophilic digestion. Of these, the most efficient reduction in infectious virus titer was achieved by mesophilic digestion with subsequent consolidation, although a pilot-scale aerobic-thermophilic digester was extremely time effective, producing sludges with similarly low virus titers in a small fraction of the time. Although none of the treatments examined consistently produced a sludge with undetectable virus levels, mesophilic digestion alone was found to be particularly unreliable in reducing the levels of infectious virus present in the raw sludge.     

Enumeration of potentially pathogenic bacteria from sewage sludges.

To ascertain the health risks that may be posed by the land application of sewage sludges, a scheme was devised to determine the types and numbers of pathogenic and potentially pathogenic bacteria present in sludges. A processing treatment was adapted to sludge to give a homogenate which yielded the greatest numbers of viable bacteria. Conventional methods were successful in enumerating Klebsiella, Staphylococcus, gram-negative enteric bacteria, and commonly used indicator organisms. Modifications of conventional methods improved the enumeration of Salmonella, Mycobacterium sp., fluorescent Pseudomonas sp., and Clostridium perfringens. However, Shigella methodology yielded only one isolate. Utilizing the proposed scheme, the population densities of these organisms were estimated in three domestic wastewater sludges. In light of these results, the potential impact of land application of sewage sludges is discussed.     

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.     

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.     

Enumeration of potentially pathogenic bacteria from sewage sludges

To ascertain the health risks that may be posed by the land application of sewage sludges, a scheme was devised to determine the types and numbers of pathogenic and potentially pathogenic bacteria present in sludges. A processing treatment was adapted to sludge to give a homogenate which yielded the greatest numbers of viable bacteria. Conventional methods were successful in enumerating Klebsiella, Staphylococcus, gram-negative enteric bacteria, and commonly used indicator organisms. Modifications of conventional methods improved the enumeration of Salmonella, Mycobacterium sp., fluorescent Pseudomonas sp., and Clostridium perfringens. However, Shigella methodology yielded only one isolate. Utilizing the proposed scheme, the population densities of these organisms were estimated in three domestic wastewater sludges. In light of these results, the potential impact of land application of sewage sludges is discussed.     

Some properties of methylotrophic bacteria isolated from sewage sludges derived from mechanical and biological sewage treatment plants.

The presented studies were carried out to get more information about physiological properties of methylotrophic bacteria selected from sewage sludges derived from mechanical and biological sewage treatment plants. All the isolated bacterial strains belonged to facultative methylotrophs. The majority of them utilized glucose, starch and lipids. Moreover, most of them were also found to possess proteolytic properties and were able to hydrolyze urea but they were incapable of asparagine ammonification.     

Isolation of indigenous enteroviruses from chemically treated and dewatered sludge samples

Samples of wastewater sludge were examined for infectious enteroviruses before and after they had been chemically conditioned and dewatered. The least virus was recovered from the cake produced by filter pressing of sludge, which had a greatly increased solids content (39 to 45% [wt/vol]) relative to the untreated sludge (4.2 to 6.2% [wt/vol]) and in one plant was at pH 11 due to the lime conditioner used. Conditioning with a cationic polyelectrolyte before dewatering by centrifugation produced a watery sludge (2.7 to 5.3% [wt/vol]) from which high titers of infectious virus were recovered which were often greater than those isolated from the untreated sludge (0.6 to 1.4% [wt/vol]). This was thought to be due to saturation of virus and sludge floc adsorption sites by the polyelectrolyte, resulting in the liberation of virions from the sludge solids.     

Fungal degradation of anaerobically digested sewage sludge

Fifty isolates of fungi were screened for their ability to grow on and degrade sludge. Cunninghamella elegans was selected for further study of degradation as affected by moisture, nutrients, time and temperature. Maximal sludge degradation (total dry weight basis) was 5.8% by Rhizopus oligosporus, 5.4% by C. elegans and 5.3% by Myrothecium verrucaria, representing approximately 11% degradation of the organic matter present. Added nutrients had little or no effect on sludge degradation. Maximal sludge degradation by C. elegans occurred in three weeks at 30–35 C or four weeks at 25 and 40 C.Sewage sludge is a stable humic material that breaks down slowly with time when mixed with soil. The disposal of sewage sludge is one of the problems of environmental pollution throughout the world. Approximately 455,000 cubic meters of sewage sludge are produced daily in the United States (7). The development of supplementary methods of sewage treatment to be used in tandem with anaerobic digestors are of current interest. Benefits of such a tandem system utilizing fungi would be the acceleration of recycling of waste, supplemental detoxification of sludge, and perhaps even recovery of utilizable protein. The use of fungi to accelerate sludge degradation with the possible side benefits of production of protein or other recoverable metabolites has not been investigated. This paper reports the screening of fifty fungi for their ability to grow on and degrade anaerobically digested sewage sludge and the determination of the level of protein produced by C. elegans on this medium.Portions of this investigation were submitted to Auburn University by W.M.H. in partial fulfillment of the requirements of the M.S. degree.Research and Technology of the U.S. Department of the Interior through the Water Resources Research Institute of Auburn University Project A-030-Ala.     

Comparison of methods for recovering indigenous viruses from raw wastewater sludge.

Five general methods for recovering indigenous viruses from raw wastewater sludge were compared. Each method included elution, concentration, and disinfection steps. The elution method, found to consistently yield the greatest viral recovery, was a two-phase technique that involved blending sludge with Freon. Other methods, including two being tested as American Society for Testing Materials tentative standard methods, were less effective. Viral recoveries were generally greater (sometimes much greater) if samples were concentrated by high-speed centrifugation rather than by organic flocculation with 3% beef extract. Three cell lines were used to measure viral recoveries by the plaque assay. The efficiency of recovery was greatest on BGM cells, followed by RD and MA-104 cells.     

Concentration of enteroviruses from large volumes of tap water, treated sewage, and seawater.

Methods are described for the efficient concentration of an enterovirus from large volumes of tap water, sewage, and seawater. Virus in acidified water (pH 3.5) in the presence of aluminum chloride was adsorbed to a 10-inch (ca. 25.4 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in a series at flow rates of up to 37.8 liters (10 gallons) per min. Adsorbed viruses were eluted from the filters with glycine buffer (pH 10.5 to 11.5), and the eluate was reconcentrated by using a combination of aluminum flocculation followed by hydroextraction. With this procedure, poliovirus in large volumes of tap water, seawater, and sewage could be concentrated with an average efficiency of 52, 53, and 50%, respectively. It was demonstrated that this method is capable of detecting surface solid-associated viruses originating from sewage treatment plants. No difference in virus recovery between laboratory batch studies and a set-up with acid-salt injection was found. This unified scheme for the concentration of viruses has many advantages over previously described systems. These include: high operating flow rates, low weight and small size, effectiveness with a variety of waters with widely varying qualities, and filters with a high resistance to clogging.     

Concentration of enteroviruses from large volumes of tap water, treated sewage, and seawater.

Methods are described for the efficient concentration of an enterovirus from large volumes of tap water, sewage, and seawater. Virus in acidified water (pH 3.5) in the presence of aluminum chloride was adsorbed to a 10-inch (ca. 25.4 cm) fiberglass depth cartridge and a 10-inch pleated epoxy-fiberglass filter in a series at flow rates of up to 37.8 liters (10 gallons) per min. Adsorbed viruses were eluted from the filters with glycine buffer (pH 10.5 to 11.5), and the eluate was reconcentrated by using a combination of aluminum flocculation followed by hydroextraction. With this procedure, poliovirus in large volumes of tap water, seawater, and sewage could be concentrated with an average efficiency of 52, 53, and 50%, respectively. It was demonstrated that this method is capable of detecting surface solid-associated viruses originating from sewage treatment plants. No difference in virus recovery between laboratory batch studies and a set-up with acid-salt injection was found. This unified scheme for the concentration of viruses has many advantages over previously described systems. These include: high operating flow rates, low weight and small size, effectiveness with a variety of waters with widely varying qualities, and filters with a high resistance to clogging.     

Seasonal distribution of enteroviruses and adenoviruses in domestic sewage

A seasonal distribution of enteroviruses and adenoviruses in raw sewage effluents of Athens, Greece, was observed over a 15-month surveillance period. All 36 samples tested were positive for both virus groups. Adenovirus concentration levels ranged from 70 to 3200 cytopathic units per litre of sample, whereas the corresponding values for enteroviruses were 90-900 cytopathic units per litre. Peak values for adenoviruses were recorded during the months of April and June 1983, whereas for enteroviruses the peak was recorded in September 1983. All three types of poliovirus were present. Coxsackievirus types B-1, B-2, B-4, and B-5 and echovirus type 7 were also isolated. Adenovirus types 1, 2, 3, 5, 7, and 15 were detected as well.     

Indexes of assessing N availability in sewage sludges

Biological and chemical methods were used in an attempt to estimate N availability in sewage sludges. The two biological methods, i.e. maize plants grown in pots, and soil-sludge mixtures incubated at 2, 4, 6, 8, 12 and 16 weeks, and the four chemical methods, i.e. autoclave, 0.5 M KMnO₄, pepsin and 0.6 M HCl, were compared to determine N availability in twelve sewage sludges in a given soil. In the mineralization test, the aerobically treated sewage sludges gave higher mineralization rates than the anaerobically treated wastes. The simple correlation between available N, estimated from the plant N uptake during 6 weeks and N extracted by chemical methods showed that HCl and pepsin appeared to be the better single indexes. Prediction of availability of N in sewage sludges to plants in the growth chamber improved if N mineralized during the incubation period and extracted by several chemical methods were combined in a multiple regression analysis.     

Production of pyrolytic liquids from industrial sewage sludges in an induction-heating reactor

With the application of induction-heating, the pyrolytic experiments have been carried out for three sewage sludges from the food processing factories in an externally heated fixed-bed reactor. The thermochemical characteristics of sludge samples were first analyzed. The results indicated that the calorific value had about 15 MJ/kg on an average, suggesting that it had a potential for biomass energy source. However, its nitrogen concentration was relatively high. From the thermogravimetric analysis (TGA) curves, it showed that the pyrolysis reaction can be almost finished in the temperature range of 450-750 degrees C. The yields of resulting liquid and char products from the pyrolysis of sewage sludge were discussed for examining the effects of pyrolysis temperature (500-800 degrees C), heating rate (200-500 degrees C/min), and holding time (1-8 min). Overall, the variation of yield was not so significant in the experimental conditions for three sewage sludges. All results of the resulting liquid products analyzed by elemental analyzer, pH meter, Karl-Fischer moisture titrator and bomb calorimeter were in consistence with those analyses by FTIR spectroscopy. Furthermore, the pyrolysis liquid products contained large amounts of water (>73% by weight) mostly derived from the bound water in the biosludge feedstocks and the condensation reactions during the pyrolysis reaction, and fewer contents of oxygenated hydrocarbons composing of carbonyl and nitrogen-containing groups, resulting in low pH and low calorific values.     

Comparison of four eluents in the recovery of indigenous viruses from raw sludge.

The efficiency of 3% casein hydrolysate (CH), 3% lactalbumin hydrolysate (LH), 3% beef extract (BE), and 10% fetal calf serum (FCS) was compared for the recovery of viruses from raw sludge. CH and LH proved to be inefficient and were eliminated from the study after initial testing. In tests with 20 different samples of raw sludge, beef extract eluted virus in 15 (75%) and FCS revealed virus in 19 (95%) of the samples using BS-C-1 cells. That different eluents were not eluting different viruses from the same sample was shown by the serologic and electron-microscopic examination of 43% (18/42) of the isolates. The identified viruses included members of the entero- (coxsackie B, and polio) and reo-virus groups.     

Biological testing of a digested sewage sludge and derived composts

Aiming to evaluate a possible loss of soil habitat function after amendment with organic wastes, a digested sewage sludge and derived composts produced with green residues, where biologically tested in the laboratory using soil animals (Eisenia andrei and Folsomia candida) and plants (Brassica rapa and Avena sativa). Each waste was tested mimicking a field application of 6ton/ha or 12ton/ha. Avoidance tests did not reveal any impact of sludge and composts to soil biota. Germination and growth tests showed that application of composts were beneficial for both plants. Composts did not affect earthworm's mass increase or reproduction, but the highest sludge amendment revealed negative effects on both parameters. Only the amendment of composts at the highest dose originated an impairment of springtails reproductive output. We suggest that bioassays using different test species may be an additional tool to evaluate effects of amendment of organic wastes in soil. Biological tests are sensitive to pollutants at low concentrations and to interactions undetected by routine chemical analysis.     

Characterization of bio-oil from induction-heating pyrolysis of food-processing sewage sludges using chromatographic analysis

In this study, gas chromatography–mass spectrometry (GC–MS) was used to analyze the pyrolytic bio-oils and gas fractions derived from the pyrolysis of industrial sewage sludges using induction-heating technique. The liquid products were obtained from the cryogenic condensation of the devolatilization fraction in a nitrogen atmosphere using a heating rate of 300 °C/min ranging from 25 to 500 °C. The analytical results showed that the pyrolysis bio-oils were very complex mixtures of organic compounds and contained a lot of nitrogenated and/or oxygenated compounds such as aliphatic hydrocarbons, phenols, pyridines, pyrroles, amines, ketones, and so on. These organic hydrocarbons containing nitrogen and/or oxygen should originate from the protein and nucleic acid textures of the microbial organisms present in the sewage sludge. The non-condensable devolatilization fractions were also composed of nitrogenated and oxygenated compounds, but contained small fractions of phenols, 1H-indoles, and fatty carboxylic acids. On the other hand, the compositions in the non-condensable gas products were principally carbon dioxide, carbon monoxide and methane analyzed by gas chromatography–thermal conductivity detector (GC–TCD).