FATE OF VIRUSES DURING AEROBIC DIGESTION
OF WASTEWATER SLUDGES
PHILLIP ROBERT SCHEUERMAN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
This dissertation is dedicated to my father in memory of my
mother, whose caring for others and love of all things
living will always be my inspiration.
The author would like to thank the members of his
committee, Dr. G. Bitton, Dr. S. R. Farrah, Dr. S. G. Zam,
Dr. T. L. Crisman, and Dr. J. E. Zoltek for their assistance
and guidance. The author would especially like to thank the
chairman of the committee, Dr. G. Bitton, and the cochairman
Dr. S. R. Farrah, for their patience, encouragement, and the
many valuable lessons. Without their help this study would
have never been completed.
The author would like to thank his friends, fellow
graduate students, and co-workers, whose support and
friendship can never be properly repaid. Special thanks are
given to Dr. Oscar Pancorbo for his assistance during this
study, to R. J. Dutton and Judy Awong for their friendship-
and strong support, to Patty Shields for her friendship and
excellent technical assistance, and to J. Bossart and J.
Kosik who helped make the Waldo project enjoyable and
successful despite the many problems.
This work was supported by grant No. R806290 from the
United States Environmental Protection Agency.
Finally, the author would like to thank his sister
Diane for her friendship and support, his sister Debbie for
her friendship, support, and her tireless help in preparing
this dissertation, and his father for his support not only
in academic endeavors but also in all other pursuits.
TABLE OF CONTENTS
ABSTRACT ................................ ................ vii
I INTRODUCTION......... .............. .... .. ..... 1
Sludge Treatment Processes................. 2
Potential Health Hazards Associated
with Sludges........................ ... .. 9
Health Risks Associated with Heavy Metals.. 10
Health Effects of Toxic Organic
Chemicals in Sludges..................... 11
Potential Health Threats from
Biological Pathogens in Sludge............ 12
University of Florida Aerobic
Sludge Project.................. ....... 20
II GENERAL MATERIALS AND METHODS.................. 22
Preparation of Virus Stocks................ 22
Standard Plaque Assay..................... 22
Assay of Indigenous Viruses by
Cytopathic Effect (CPE) ................. 24.
Assay of Indigenous Viruses by
Plaque Assay. ............ ...... ......... 25
Typing of Indigenous Isolates............... 25
Preparation of Bacteriophage Stocks........ 26
Bacteriophage Assays...................... 27
Sludges and Sludge Sources................. 28
III LABORATORY STUDIES OF VIRUS SURVIVAL DURING
AEROBIC AND ANAEROBIC DIGESTION OF
SEWAGE SLUDGE........... ............... ......... 31
Literature Survey.......................... 33
Materials and Methods...................... 37
Results and Discussion.................... 47
IV DEVELOPMENT OF METHODOLOGY FOR RECOVERY OF
VIRUSES FROM AEROBIC SLUDGES................... 71
Introduction................... ............ 71
Literature Survey............... ............. 71
Materials and Methods..................... 81
Results and Discussion..................... 90
Conclusions... ...... ...... ... .......... .. 112
V MONITORING OF INDIGENOUS ENTEROVIRUSES IN
AEROBICALLY DIGESTED SLUDGES AT TWO WASTEWATER
TREATMENT PLANTS IN GAINESVILLE, FLORIDA........ 113
Literature Survey .......................... 114
Materials and Methods..................... 119
Results and Discussion..................... 120
VI FURTHER LABORATORY STUDIES ON THE SURVIVAL OF
VIRUSES IN AEROBIC SLUDGES..................... 153
Introduction................. .............. 153
Literature Survey. ........................ 154
Materials and Methods...................... 157
Results and Discussion..................... 164
Conclusions.......................... ....... 197
VII CONCLUSIONS... .. ...... ........ ........ ........ 198
APPENDIX: COMPOSITION OF MEDIA AND SOLUTIONS......... 202
BIBLIOGRAPHY........ ..... .............. ............... .. 207
BIOGRAPHICAL SKETCH................................... 215
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
FATE OF VIRUSES DURING AEROBIC DIGESTION
OF WASTEWATER SLUDGES
Phillip Robert Scheuerman
Chairman: Gabriel Bitton
Cochairman: Samuel R. Farrah
Major Department: Environmental Engineering Sciences
The concern for protection of water bodies and public
health has led to the search for treatment and disposal
practices for wastewater sludges which will minimize
pollution and health threats. Aerobic stabilization of
wastewater sludges is a popular alternative to anaerobic
digestion, especially in warm climate areas such as Florida.
However, little is known about the removal of pathogens
during this process. The objective of this study was to
assess the effectiveness of aerobic sludge digestion to
The survival of virus during aerobic stabilization of
wastewater sludges was studied using laboratory scale
digesters. A laboratory scale anaerobic digester was used
for comparison. Survival of poliovirus type 1 (Lsc) during
aerobic digestion was found to be lower than during
anaerobic digestion. Temperature was found to play a major
role in the rate of inactivation, with increased survival as
the temperature was decreased from 280C to 5 OC. The extent
of survival was not found to vary with virus type, dissolved
oxygen concentration, detention time and sludge source.
Seven methods were compared for recovery of viruses
from aerobic sludges, with the trichloroacetic acid-lysine
method chosen as the method for further studies. The
survival of indigenous viruses and indicator bacteria was
studied in sludges at two local wastewater treatment plants
using the aerobic digestion process. Viruses were
determined as TCID50/g and PFU/g. Virus levels at both
plants were reduced more than 90% during two-stage
digestion. Parameters such as temperature, pH, total
solids, volatile solids, dissolved oxygen, and conductivity
were evaluated for correlation with TCID50/g and PFU/g.
Factors which correlated significantly varied with sludge
type, treatment plant, and assay method.
Fractioning the sludge into its component parts (solids
and liquid) indicated that virus survival was decreased
after removal of the sludge flocs. Attempts to isolate an
antiviral agent in the sludge liquor were negative. The
presence of a protective component which was neutralized or
removed by some aerobic process was proposed as an
explanation for the greater removal during aerobic as
opposed to anaerobic digestion.
Evaporative drying of aerobic sludges was found to
reduce the number of viruses recovered.
Waste disposal has plagued man throughout history.
With increased population growth and industrialization the
quantity and quality of wastewater has undergone severe
changes. The added load on receiving waters due to increase
in volume and level of recalcitrant compounds has resulted
in a decline in water quality. The increased volumes have
begun to overload receiving water bodies, reducing the
effect of dilution and natural purification processes. In
addition many compounds are not degradable in the
environment and subsequently accumulate in water and
sediments. These compounds may not only affect the esthetic
quality of the water but may actually pose a health threat
to humans and livestock using this water. This results in
expensive processes required to treat the water to meet
requirements necessary for potable use.
In answer to these problems the United States Congress
passed the Federal Water Pollution Control Act Amendments of
1972 (P.L. 92-500) which called for significant water
quality improvements by 1983 and elimination of pollutant
discharge into navigable surface waters by 1985. Congress
further strengthened this mandate by passing the Clean Water
Act of 1977. Section 405 of the Clean Water Act requires
that regulations be issued which provide guidelines for the
disposal and utilization of sludge. Herbert Pahren (1980)
of the United States Environmental Protection Agency has
stated that the goal of the national sludge management
program is to utilize available resources while at the same
time reducing public health risks to an acceptable level.
Sludge Treatment Processes
Wastewater can be treated using various physical,
chemical, and biological unit processes which may be used in
various combinations. The most basic process, designated as
primary treatment, removes settleable solids from wastewater
and thus makes the water less objectionable and easier to
treat in subsequent unit processes (Vesilind 1979, Clark et
al., 1971). This treatment is carried out in the primary
clarifier which operates by allowing heavier solids to
settle to the bottom and lighter solids to float to the top.
The floating material, which is often termed scum, is
usually not treated further, but is disposed of with other
waste solids. The raw primary sludge is collected at the
bottom and requires further treatment prior to disposal.
Secondary treatment is designed to remove both solids
and BOD (Biochemical Oxygen Demand). Secondary treatment is
performed using biological processes. The two most common
are trickling filters and the activated sludge process. The
trickling filter process involves the passage of wastewater
over a filter bed of rocks or other such media. The filter
media is covered with a film of microorganisms which
performs the biological removal of organic matter.
Periodically this mass of organisms will slough off
producing solids which are recovered in the final clarifier.
The second means of secondary treatment is the activated
sludge process. In this process the microorganisms and the
wastewater are mixed in a large tank and air or oxygen is
introduced into this mixture by some form of diffuser or
mechanical surface aerator. The biomass produced in the
activated sludge process grows and produces more organisms
than are required to perform the organic removal. The
excess biomass is consequently wasted (waste activated
sludge) and will require stabilization prior to disposal.
It can be seen that during the treatment of municipal
wastewater large quantities of solid particles are produced
and consequently require removal. These particles are
usually removed by settling, producing large amounts of
materials termed sludges. Sludge handling and disposal is a
major part of wastewater treatment. From 25-40% of the
total cost of a plant is invested in sludge-processing
equipment, and much of the normal plant operation and
maintenance is concerned with sludge disposal (Clark et al.,
1971). During municipal wastewater treatment an average of
35% of the influent BOD becomes waste activated sludge
(Benefield and Randall, 1980).
The portion of the U.S. population served by sewers was
estimated to be 67% of the population in 1970 and is
projected to increase to 75% in the mid 1980'S (CAST, 1976).
Sludge production per person is estimated at 0.055 to 0.091
kg/day dry weight, depending on the treatment process used.
Implementation of advanced wastewater treatment increases
sludge production by additional removal of suspended and
dissolved solids. This along with the projected increase in
population and percent of population on municipal sewers
should lead to a marked increase in sludge production..
Indeed the quantity of sludge produced on a yearly basis
was 3.6 million metric tons in 1970 (CAST,1976) and 4.5
million metric tons in 1979 (Pahren et al., 1979). This
quantity is projected to be from 7.3 to 8 million metric
tons/year in the 1980's (CAST, 1976, Pahren et al., 1979).
The distribution of sludge disposal methods was
estimated in 1979 to be 35% incineration, 15% ocean
disposal, 25% landfill, and 25% land application (Pahren et
al., 1979). The banning of ocean disposal and air pollution
problems associated with incineration led to an increase in
land disposal to at least 65% in the 1980'S. Using the
projection of 7.3 million metric tons/year produced, the
quantity disposed on land will be approximately 4.7 million
metric tons/year in the 1980'S. Clearly this indicates that
a strong management program for sludge treatment and
disposal is required.
The final disposal of sludges can be performed
following treatment of the sludge using one of several
steps: sludge stabilization, sludge thickening, sludge
conditioning and sludge dewatering. The goal of these
processes is generally to minimize the sludge volume, remove
the excess water, and produce a less offensive final product
(EPA, 1975). These various processes used to treat sludges
are listed in Table 1-1. Many of these treatment processes
are used in various combinations, while others are used
independently (Clark et al., 1971; Benefield and Randall,
1980; EPA, 1974).
The primary requirements of sludge stabilization are
the removal or reduction of the highly volatile portion of
the sludge in order to reduce odor problems, remove or
transform toxicants to a form less likely to adversely
Table 1-1. Common methods used for sludge
treatment and disposal.
1. Anaerobic digestion
2. Aerobic digestion
C. PHYSICAL METHODS
1. Sludge thickening
2. Sludge conditioning
3. Sludge dewatering
a. Sand beds
b. Vacuum filtration
d. Filter press
e. Belt filters
D. FINAL DISPOSAL
2. Wet oxidation
3. Land Spreading
4. Landfill of sludge
5. Landfill of incineration ash
1975 and Clark et al., 1971.
affect the environment or cause a public health threat, and
result in the maximum possible kill of pathogenic organisms
(EPA, 1975). Traditionally, sludge stabilization has been
accomplished by anaerobic digestion (Benefield and Randall,
1980). However, problems such as high nutrient and organic
matter content of the sludge supernatant and the high
sensitivity of the anaerobic process to perturbation have
increased the popularity of alternate treatment processes
such as the aerobic digestion process.
Anaerobic digestion can be performed using either one
of two processes; standard-rate digestion or high-rate
digestion. The standard rate process is performed in a
single digester without mixing, allowing the digester
contents to stratify into zones. The digester is generally
fed sludge and withdrawn intermittently. This process
requires long detention times which are shortened by heating
to increase the digestion rate. According to Benefield and-
Randall (1980) the required detention time is 30-60 days
and the organic loading rate is between 0.03 Ib and 0.1 Ib
total volatile solids per cubic foot of digester volume per
The high-rate process uses two digesters in series.
The first is completely mixed by gas recirculation or
mechanical mixing and the sludge is heated. The purpose of
this stage is to carry out the fermentation process. The
second stage is designed to achieve solids-liquid separation
and is therefore unmixed and generally not heated. Sludge
feeding and withdrawal is continuous. The retention time is
10-15 days with 0.1-0.4 Ib total volatile solids per cubic
foot of digester volume per day organic loading (Benefield
and Randall, 1980). Volatile solids are reduced 40-60%
The aerobic digestion of biological solids is nothing
more than a continuation of the activated sludge process
(Benefield and Randall, 1980). Aerobic digesters are being
used to stabilize sewage sludge more often because of their
ease of operation compared with that of anaerobic digesters
(EPA, 1975). In addition aerobic digesters are not as
sensitive as anaerobic digesters to sudden changes in
temperature and wastewater composition (Benefield and
Randall, 1980). Another important advantage is the lower
BOD found in supernatant liquors from aerobic digesters
versus that of the sludge liquor from anaerobic digesters.
However, aerobic digesters are more expensive to operate due
to the aeration required. Aerobic digestion can be
performed in a single digester or a series of digesters to
better approximate plug flow conditions and achieve higher
removal efficiencies. Aeration periods of 10 days at 200C
and 15 days at 100 are generally adequate and organic
loadings should be less than 0.07 lb total volatile solids
per cubic foot of digester volume per day. The volatile
solids reduction falls in the range of 30-55%.
Potential Health Hazards Associated with Sludges
Earlier in this chapter Herbert Pahren (1980) was
quoted concerning the disposal of sludges and the need to
reduce the associated public health risks. These public
health risks include toxic metals, trace organic, and
The potential health effects from sludge disposal are a
serious concern. However, the magnitude of this health
threat is not known. The presence alone of toxic chemicals
and pathogens does not indicate that a health threat does
indeed exist. The concentration required to cause these
health problems must be determined. It is also important to
determine the fate of these health-threatening components
after disposal, i.e., do they concentrate and consequently
build up to health threatening concentrations or are they
rendered harmless by various reactions during sludge
stabilization and disposal?
Health Risks Associated with Heavy Metals
The potential for health effects from toxic metals in
sludges is generally agreed upon to be most significant with
cadmium (Chaney, 1980; Lucas et al., 1977; and Pahren et
al., 1979). While the potential from other metals does
exist it is in relatively isolated and controllable cases.
Copper toxicity is probably of little risk to humans
and livestock, since in cases where copper concentrations
are high enough to pose a potential health threat, severe
phytotoxicity occurs (Lucas et al., 1977 and Pahren et al.,
Mercury concentrations in sludges are in general low
enough to pose little health threat. In cases where mercury
is added to soil in large quantities, it may be taken up by
plants but is more likely to be bound by clay and organic
matter (Lucas et al., 1977 and Pahren et al., 1979).
Chromium is reduced to the trivalent state during
sludge digestion and consequently pose few health problems.
Molybdenum is generally not found in very significant
concentrations in sludges and therefore poses no health
threat except in very unusual circumstances. Both nickel
and zinc are found in relatively high concentrations in
sludge but both are of low toxicity to humans. Lead, tin,
manganese, iron, and aluminum are all considered to be of
little concern, the latter three due to their low solubility
(Lucas et al., 1977 and Pahren et al., 1979).
Cadmium is of concern due to its relatively high
concentrations in sludges. It is also appreciably taken up
and accumulated by plants especially the green leafy
vegetables. The main target organ in the human body is the
kidney, and Cadmium is accumulated in this organ (Lucas et
al., 1977). It is concluded however, that cadmium in
municipal sludges applied to agricultural lands will not be
a health problem. This is attributed to the marketing
practices and types of crops generally grown on sludge
amended soils (Lucas et al., 1977 and Pahren et al., 1979).
Health Effects of Toxic Organic Chemicals in Sludges
Compounds such as organochlorine insecticides,
chlorinated phenolics, polyaromatic ring compounds,.
phthalates, and sulfonates may be found in sludges and are
of concern. Most of these compounds will in general be
present in sludges because of their low water solubility.
The organochlorine compounds are especially of concern due
to their high resistance to degradation and they are
generally lipid-soluble (Dacre, 1977 and Dacre, 1980). An
example of chemical contamination of sludge which presented
a severe health threat came to light with the discovery of
PCB's in the milk of a cow grazing on pasture land that had
received 12 tons/acre of municipal sludge five months
before. Analysis of the sludge showed 105 and 240 ppm PCB's
(dry weight). In addition analysis of the cow's milk showed
5 ppm PCB's (Torrey, 1979).
Potential Health Threats from Biological Pathogens in Sludge
The incidence of disease transmission, particularly
Schistosomiasis, associated with the night soil crop
fertilization in China and the epidemics of typhoid fever in
other parts of the world attributed to the consumption of
raw vegetables irrigated with raw sewage have caused a
general aversion to the use of even treated human waste
products (Torrey, 1979). Biological pathogens in sludges
include parasites, bacteria, and viruses. Table 1-2 gives
the major biological pathogens associated with wastewater
sludges, and some of the diseases they cause. The extent
of the health hazard posed by enteric pathogens in sludge,
TABLE 1-2. Some organisms of public health significance
which may be found in wastewater sludges.
Coxsackievirus group A
Coxsackievirus group B
Congenital heart anomalies
Table 1-2. Continued
Echovirus Respiratory infection
Reovirus Respiratory infection
Adenovirus Acute conjuctivitis
Acute upper respiratory illness
Hepatitis A Infectious hepatitis
Rotavirus Infantile gastroenteritis
Norwalk agent Nonbacterial gastroenteritis
Sources: Bitton, 1980; Sagik et al., 1980; Akin et al., 1977;
and Zam S. G. (personal communication).
however, remains an unanswered question (Dawson et
al.,1981). Larkin et al. (1977) point out that due to the
inability of sewage treatment plants to produce sludges and
effluents devoid of microbial pathogens and because of
limitations in monitoring systems, -the Food and Drug
Administration recommends no food- crops that are to be
consumed raw should be grown on soils that are amended with
sludges. In addition, because of the persistence of some
parasites, sludge-treated lands should not be used for such
crops until three years after sludge application has ceased.
Protozoan and Helminth parasites are of concern due to
their ability to resist the conditions existing during
wastewater treatment. Little work has been done on
these sludge-associated organisms. However, the little
information available indicates that there are numerous.
different parasites in many stages of their life cycles
found in sludges (Akin et al., 1977; Little, 1980). Little
(1980) reported on a one year study of 27 different
treatment plants. He reported that none of the stages of
protozoa found in treated sludges appeared to be viable.
The most commonly found were the eggs of Ascaris, Toxocara,
and Trichuris. Kabrik et al. (1979) found similar results
in their study comparing the removal of various pathogens in
thermophilic aerobic digestion versus anaerobic digestion.
They also found that removal of total viable ova was
superior during aerobic digestion to that during the
anaerobic process.. Concerning the health effects of
parasites Little (1980) concluded that even though the
paucity of reports on domestic animals acquiring parasitic
infections from land application of sludges indicated little
health risk, undetected light infections could serve as a
source for dissemination of infections to other animals or
The most fragile of the four groups of pathogens found
in sewage are the bacteria. They are poor competitors
outside the host animals and in the biologically active
environment of secondary sewage treatment, their numbers are
significantly reduced (Akin et al., 1977). Studies on the
removal of bacteria during anaerobic digestion of sludges
have shown that complete removal of indicator bacteria and
the pathogenic bacteria is not achieved (Dudley et al.,
1980; Kabrick et al., 1979 ). Kabrick et al., (1979) found
that the removal of Salmonella sp. and Pseudomonas
aeruginosa during thermophilic aerobic digestion of sludges
exceeded by one to three orders of magnitude the removal
during anaerobic digestion.
Approximately 100 types of enteric viruses are present
in raw sewage (Akin et al., 1977). There have been only a
few outbreaks of enteroviral diseases attributed to
contaminated waters. Probably the most significant was an
outbreak of Hepatitis A in Delhi, India, that resulted in
approximately 30,000 cases (Bitton, 1980). A leak in a
wastewater lagoon in Missouri resulted in numerous cases of
acute gastroenteritis. This outbreak was attributed to
contamination of unchlorinated private wells with an enteric
virus (Kowal and Pahren, 1979). Table 1-3 lists several
outbreaks of hepatitis and viral gastroenteritis due to
contaminated water sources. The removal of viruses during
sludge treatment will be discussed in several other sections
of this work, and will not be covered in this introduction.
To summarize the potential health effects due to sludge
disposal it can be stated that the potential does exist
however the reality and magnitude of this threat are
questioned. Akin et al., (1977) stated that the increase in
land application of domestic waste will, without question,
increase the risk of exposure to microbial pathogens for
TABLE 1-3. Some waterborne outbreaks of Hepatitis and
Gastroenteritis since 1950.
Date of Number of
occurence Place cases Vehicle Disease
Delhi, India 28,745
New Jersey 13,000
New York 7
West Virginia 700
rom Bitton, 1980.
some segments of the population, especially the personnel
engaged in the transport and application of the waste to
land. However, as pointed out by Burge and Marsh (1978)
little evidence exists to show that disease is transmitted
by land spreading of treated sewage sludge and effluents.
Furthermore, the studies that have been done to answer some
of these questions only confuse the issue more as evidenced
by two studies reviewed by Kowal and Pahren, (1979). These
studies were aimed at determining the health threat posed to
workers and nearby residents of disposal sites. These
studies presented conflicting results, with one study
showing increased risk of respiratory and gastrointestinal
illness and the second unable to show any such risk. Akin
et al. (1977, pp. 20-21) summarized this dilemma with the
following statement: "In the absence of more effective
treatment processes and in the absence of recognized disease
transmission from the land application of treated sewage,
it would appear to be totally unrealistic to require all
domestic wastes to be pathogen-free. No such absolute
condition could be guaranteed without the complete testing
of all waste with methods that were 100 percent efficient
for all pathogens. Such a testing program would be
economically impractical and technically unattainable. We
therefore are left with the subjective goal of achieving and
maintaining the microbial hazard from waste disposal on land
at an 'acceptable risk' level." As pointed out by Torrey
(1979) the reporting of enteric disease cases is notoriously
poor, and sufficient evidence is not yet available to
discuss the epidemiological significance of complex
populations of bacteria, viruses, and parasites found in
sludge. Therefore it is important that studies continue to
obtain both the epidemiologic data necessary to judge the
health threats and the treatment information required to
minimize these health threats.
University of Florida Aerobic Sludge Project
This work is part of a larger project designed to
obtain information on the removal of biological pathogens
during aerobic stabilization of wastewater sludges. This
project studied the survival of parasitic ova, enteric
bacteria, and enteric viruses during aerobic mesophilic
digestion of sludge. This work will report on the survival
of enteric viruses during aerobic digestion. The major
objectives of this project were to
1. Determine in laboratory experiments the degree of
virus inactivation during aerobic digestion.
2. Monitor the level of indigenous virus before and
after aerobic digestion during the year.
3. Examine the inactivation of viruses for possible
operational parameters which correspond to
inactivation and may actually be controlling.
4. Examine the possible mechanisms of inactivation of
viruses as well as the sludge component responsible
for such inactivation.
This was approached through laboratory studies and
field studies. Laboratory studies were aimed at studying the
effect of operational parameters, to be discussed later, on
virus inactivation. In addition, laboratory studies were
used to evaluate other possible explanations for virus
inactivation. Field studies were used to test the results
of the laboratory studies and to look at other seasonal
trends not controllable during laboratory studies.
GENERAL MATERIALS AND METHODS
Preparation of Virus Stocks
Virus used for routine experimentation was prepared by
infecting a 32 ounce culture bottle of the appropriate host
cell line with approximately 106 PFU. After a lhr
adsorption period at room temperature, 50 ml of MEM + 2%
Fetal calf serum (FCS) and antibiotics (see appendix) was
added and the infected monolayer was incubated at 370C until
4+ CPE was observed. Cells were freeze-thawed three times
and the subsequent harvest clarified by centrifugation for
10 minutes at 16,000 X g. The clarified supernatant was
transferred in aliquots (2-3 ml) to screw capped vials and
frozen at -20 C for routine use. If prolonged storage.
before use was anticipated stocks were stored at -70 C.
Standard Plaque Assay
Virus was assayed on either BGM, Vero, or MA-104 cell
lines, all monkey kidney cells. Monolayers were prepared by
incubating cells in a 32 ounce bottle using a growth medium
consisting of Eagle's MEM + 10% FCS (see appendix) until
they reached confluent monolayers. After the monolayers were
confluent, the growth medium was decanted and the cells were
washed with 10 ml of a pre-trypsin solution (see appendix)
three times. The cells were then removed from the glass
bottles by covering them with 10 ml- of a standard trypsin-
versene solution (see appendix). After 30-60 seconds, all
but approximately 1 ml of this solution was decanted and the
monolayer was kept at room temperature until the cells began
to slough off the glass ( approximately 5 min.). Then, 10
ml of Eagle's MEM + 10% FCS were added and pipetted up and
down to dislodge the cells. Following this treatment 190 ml
of Eagle's MEM + 10% FCS were added and the suspension was
then distributed in 5 ml aliquots to 25 cm2 plastic tissue
culture flasks. After incubation for 48 hr at 370C the
tissue cultures were ready for use in virus assays. These
were performed by infecting the monolayer with 0.1-0.5 ml of.
the virus suspension which had been diluted in phosphate
buffered saline (PBS) plus antibiotics (see appendix) to
yield a countable number of 100-300 plaques per flask.
Following infection, the virus was allowed to adsorb for 1
hour with tilting at 15-minute intervals. The infected
monolayers were then covered with 5 ml of a methyl cellulose
overlay (see appendix) and incubated at 370C for a time
appropriate for the stock virus being assayed. Plaques were
visualized by the addition of 0.5 ml of 0.1% neutral red
(see appendix) to the flask at the end of the incubation
Assay of Indigenous Viruses by Cytopathic Effect (CPE)
Replicate tube or bottle cultures of BGM or Vero cells
were infected with 0.1 to 0.5 ml of sample and incubated at
room temperature for 1 hr. If toxicity or bacterial
contamination was anticipated the sample was pre-filtered
through a 0.25-3.0 :pm Filterite filter series (Filterite
Corp., Timonium, Md.) in a 25 mm holder, followed by an
equal volume of Eagle's MEM + 2% FCS and antibiotics. The
infected monolayers were drained of excess sample and washed
3X with PBS + antibiotics at the end of the 1-hr adsorption
period. Subsequent to the 1-hr adsorption period the
monolayers were supplemented with growth media (MEM + 2% FCS
and antibiotics) and incubated at 370C. The monolayers were
scored for CPE at 2-day intervals for up to 21 days with
media changes at the same intervals. All cultures showing
positive CPE were frozen, thawed and passed to fresh
cultures for confirmation. The 50% tissue culture infective
dose was determined by the procedure of Reed and Muench
Assay of Indigenous Viruses by Plaque Assay
Monolayers of BGM or Vero cells in 25 cm2 and 75 cm2
plastic culture bottles were infected with 0.2 to 1.0 ml of
sample and incubated at room temperature for 1 hour.
Subsequently, the monolayer was overlayed with Eagle's MEM
supplemented with 2% FCS, antibiotics, and 1% Bacto-Difco
agar (Difco Laboratories, Detroit Mich.). If toxicity or
bacterial contamination was anticipated samples and infected
monolayers were treated as with the CPE assay. The cultures
were then incubated at 370C for 48 hours, a second overlay
was subsequently added containing MEM, 2% FCS, 1% Bacto-
Difco agar and 0.004% neutral red. Monolayers were scored
for plaques for up to 14 days. At least 50% of the plaques
appearing were picked for confirmation. Plaques were
confirmed by passage into tube cultures and observed for CPE
for up to 21 days. If confirmation was not obtained on the
first passage a second aliquot was plated for confirmation.
Typing of Indigenous Isolates
Virus isolates from sludges were identified using
poliovirus antiserum (M.A. Bioproducts Walkersville, Md.) to
minimize the use of the antiserum pools. Subsequently, pools
of neutralizing antiserum (Lim and Benyesh-Melnick, 1960)
were used to type non-poliovirus isolates. Confirmed
isolates were purified by endpoint dilution and mixed with
equal volumes of each antiserum pool. The virus-serum
mixtures were incubated for 2 hours at 370C and then
inoculated (0.1 ml) onto BGM monolayers in microtiter
plates. After a 30-minute adsorption period at room
temperature the monolayers were overlayed with methyl
celluose (see appendix) and incubated at 370C for 48 hours.
Plaques were visualized for counting by staining at the end
of the incubation period with 0.05 ml of 0.1% neutral red.
Pools showing an 80% reduction with respect to a serum free
control were considered neutralized.
Preparation of Bacteriophage Stocks
MS-2 Bacteriophage stocks were prepared using the
procedure described by Yamamoto and Alberts (1970). The
host bacterium Escherichia coli C-3000 was innoculated from
a stock slant into 10 ml of 3% Tryptic Soy Broth (Difco
Laboratories, Detroit Mich.) and incubated overnight (18 hr)
at 370C. This overnight culture was innoculated into M9-
media (see appendix)(lml/100ml) and incubated overnight (18
hr) at 370C. Bacteriophage was subsequently innoculated at
a multiplicity of infection of 10X. The bacteriophage-
infected cultures were incubated overnight (18 hr) at 370C.
Chloroform was subsequently added (0.1 x vol.) and the
culture was agitated for 30 minutes. Filtered humidified
air was then bubbled through for 1 hr at an approximate rate
of 1 bubble/second, to remove excess chloroform. 'The
cultures were then centrifuged at 8-,000 X g for 5 minutes.
The pH of the supernatant was adjusted to 7.0 with IN NaOH
and polyethylene glycol (M.W. 6000) was added to a final
concentration of 6% (W/V). This mixture was incubated at 4 C
overnight, and subsequently centrifuged at 8,000 X g for 10
minutes. The virus containing pellet was resuspended in PBS
supplemented with 3% beef extract. This mixture was
centrifuged at 16,500 X g for 10 minutes. All bacteriophage
stocks were stored at 40C.
Bacteriophage were assayed using an agar-overlay plaque
assay. Host stocks were prepared by innoculating 10 ml of
3% tryptic soy broth from a stock nutrient agar slant and
incubated overnight (18 hr) at 370C. This overnight culture
was subsequently diluted 1:10 into a second aliquot of 3%
tryptic soy broth and incubated at 370C for approximately 4
Bacteriophage samples were diluted in PBS supplemented
with 3% beef extract pH 7.2. To 4 ml of molten agar (see
appendix) was added 0.1 ml of phage dilution and 0.2 ml host
bacterium. This mixture was poured into a petri dish
containing approximately 20 ml of base agar (see appendix),
allowed to solidify, and incubated overnight (18 hr) at
Sludges and Sludge Sources
Sludges used in this study and their sources are
described in Table 2-1. The majority of the sludges used in
this study were obtained from three treatment plants:
Gainesville Kanapaha, Gainesville Main Street, and the
University of Florida campus plant.
The Gainesville Kanapaha plant is an advanced secondary
treatment plant using activated sludge and denitrification.
Sludges are treated using two 8.5 x 105 gallon aerobic
digesters in series with a detention time of 8.5 days each.
Liquid digested sludges are spread on a "sludge farm"
operated by the City of Gainesville.
The Gainesville Main Street plant is a secondary
treatment plant using both trickling filters and activated
sludge. Sludges are treated in two 1.3 x 106 gallon aerobic
digesters in series with a detention time of 50 days each.
Table 2-1. Treatment characteristics of sludges used in this study.
Sludge Sludge time Additional
Plant designation* type (days) treatment
Campus plant UML Mixed liquor ---
(University of UDA Aerobically 39
Gainesville Fl. GW Waste** 25+
Main Street GML Mixed liquor ---
GDA 50 Aerobically 50
GDA 100 digested 100+
Gainesville Fl. KW Waste 30+
Kanapaha KML Mixed liquor ---
KDA 8.5 Aerobically 8.5
KDA 17 digested 17++
Pensacola Fl. PDA Aerobically 30 Conditioned
Montclair digested with Magnifloc
(dewatered) 1563C and then
Pensacola Fl. PDAN Anaerobically 60 Conditioned
Main Street digested with Magnifloc
(dewatered) 2535C and then
Montclair and LAG Lagooned ---
* These abbreviations will be used throughout the rest of this study.
** Equal volumes of waste activated and primary sludge.
+ Sludge age.
+ Total detention time, includes stage-1.
The Main Street plant is unusual in that it has the
long detention time not normally associated with aerobic
digestion. The reason for this long detention time is that
approximately 50% of the sludge fed to the digesters is
primary sludge. This also poses a problem in analyzing the
removal rates in the first stage digester because it is not
possible to analyze the concentration of viruses entering
the digesters due to lack of any sampling point. Liquid
digested sludges are disposed on the same disposal site as
those from the Gainesville Kanapaha plant.
The University of Florida campus treatment plant is a
secondary treatment plant using both activated sludge
(contact stabilization) and trickling filters. Sludges are
treated using 3 to 4, depending upon flow, aerobic digesters
in series with a 13 day detention time each. Sludges are
dewatered on drying beds.
LABORATORY STUDIES OF VIRUS SURVIVAL DURING
AEROBIC AND ANAEROBIC DIGESTION OF SEWAGE SLUDGE
The use of laboratory scale digesters to study which
operational parameters may have the greatest effect upon
virus survival is described in this chapter. Laboratory
studies allow for controlled conditions under which these
effects can be easily studied. The parameters which will be
studied are listed in Table 3-1. However, it should be
remembered that laboratory scale experiments will not
completely reflect the complex interactions which occur in
full scale digesters at the wastewater treatment plant.
Many factors cannot be accurately duplicated or simulated-
when scaling down the operation. Many factors such as the
method of feed and draw of sludge are not performed the same
way in the field and in the lab. In most laboratory
experiments the sludge is fed and withdrawn once during the
detention period; however, during operation of full scale
digesters sludge may be drawn and added several times during
the day in quantities sufficient to achieve the desired
detention period. Sludge is often fed to the digester just
Table 3-1 Digestion conditions which will be studied.
Secondary Total solids
Hydrogen ion (10-pH)
* Primary parameters were controlled at set levels for
each run, secondary parameters were monitored for
prior to the drawdown with the result that a portion of the
fresh sludge is drawn off with the digested sludge. In
addition, the laboratory scale digester is generally more
thoroughly mixed than the full scale digester. Many
operational conditions such as temperature are more
controlled in the laboratory and consequently fluctuations
are less prevalent in the laboratory. Generally the
laboratory and pilot scale digesters are maintained in
optimal working conditions with little down time while full
scale digesters may not be operating at peak efficiency and
may suffer from frequent down periods. Consequently, the
optimal conditions available in the laboratory are not often
obtained in the full scale digesters and therefore one
should examine these laboratory studies with the purpose of
observing trends and not actual removal rates.
A search of the literature reveals that no laboratory
studies on the survival of viruses following aerobic
digestion of sludge have been performed. However, Kelly et
al. (1961) studied the removal of poliovirus and T2
bacteriophage from activated sludge. The activated sludge
process is operationally similar to aerobic sludge digestion
(see Chapter 1) and consequently some comparison can be made
between these two processes. They found that over a 30-day
period the removal of added virus involved at least two
steps, aeration in the presence of sludge floc and nutrient,
and the settling of floc. Aeration of raw sewage did not
reduce virus or phage concentration. However, when the
mixed liquor was aerated for different periods of time, the
longer aeration resulted in greater removal. Viruses were
found to be inactivated during the solids removal process.
Bacteria with antiviral activity were isolated, suggesting
biological antagonism as a possible third step.
The survival of viruses during anaerobic digestion has
been extensively studied in the laboratory. Consequently,
much more is understood about the virus removal efficiency
and the possible mechanism associated with this process.
Bertucci et al. (1975 and 1977) studied the survival
of four enteric viruses (poliovirus 1, coxsackievirus A9,
coxsackievirus B4, and echovirus 11) and one bacteriophage.
(MS2). They found inactivation rates of 75%, 90%, 97%, 90%,
and 93% for echovirus 11, coxsackievirus B4, coxsackievirus
A9, poliovirus 1, and bacteriophage MS2, respectively.
These rates were found to correspond to three distinct
groups at the 95% confidence level with group 1 consisting
of echovirus 11, group 2 the bacteriophage MS 2, poliovirus
1, coxsackievirus B4 and group 3 coxsackievirus A9. They
also noted that the inactivation appeared to follow a first
order reaction equation.
Eisenhardt et al. (1977) studied the survival of
coxsackievirus B3 in a pilot scale anaerobic digester. They
found that coxsackievirus was reduced approximately 0.4
log/d at 320C and pH 7.0, and 1.5 log/d at 350C and pH 7.0.
Sanders et al. (1979) studied the survival of
poliovirus type 1 at 340C and 370C in laboratory scale
anaerobic digesters. Poliovirus was introduced into aerated
waste activated sludge, which was subsequently introduced
into 2-liter bench scale anaerobic digesters. Poliovirus
was inactivated at two different rates, an initial rapid
rate of 84 to 99%/day for the first 24 hours. The second
rate was 30-60%/day from 24 hours until the end of the run
or until virus was no longer detectable. They found that
temperature had a significant effect on the loss of virus in
both controls and digesters. They also found that detention
time played a decreasing role as the digestion temperature
was increased. Virus survival was found to decrease as
total and volatile solids increased.
Subrahmanyan (1977) added several virus types to
sewage sludge which was stored at room temperature and
tested for viruses at intervals for up to 12 weeks.
Coxsackieviruses were found to be among both the most
resistant and the least resistant, coxsackievirus A9 was the
least resistant and coxsackievirus B5 along with echovirus 6
were the most resistant. Coxsackievirus A9 survived less
than two weeks, coxsackievirus B3 survived 4 weeks,
coxsackievirus B2 5 weeks, poliovirus 1 (vaccine) 8 weeks,
poliovirus 1 (virulent) 10 weeks, poliovirus 3 (virulent)
less than 8 weeks, echovirus 6 was still detectable at 12
weeks, coxsackievirus B5 was still detectable at 12 weeks,
and reovirus 2 survived 6 weeks.
Ward and Ashley (1976, 1977A, 1977B, 1977C, 1978A) and
Ward et al. (1976) studied the survival of virus in
anaerobically digested sludge. As did Sanders et al., 1979,
they found an initial rapid inactivation of poliovirus in
anaerobically digested sludge. Inactivation of poliovirus
in the absence of sludge was 1 log/5 days at 280C and not
detectable at 200C or 5*C. In the presence of sludge
poliovirus was inactivated at a rate greater than 1 log/day
at 280C and 1 log/5 days at 40C. Raw sludge was protective
of poliovirus at 430C, 47'C, and 510C while digested sludge
accelerated inactivation at 510C. At all temperatures
inactivation was greater in digested sludge than in raw
sludge. The protective component of raw sludge was found to
be retained after digestion, at higher sludge concentration
and temperatures. However, the expression of the
protective component was diminished by another substance
found only in digested sludge. Ammonia was identified as the
virucidal agent in digested sludges. It was also found that
the detergents that were protective of poliovirus were the
same agent which accelerated the heat inactivation of
reovirus. This work will be discussed in more detail in
Materials and Methods
Viruses and Viral Assays
Viruses used for digester experiments were poliovirus
1 (strain Lsc), coxsackievirus B3, echovirus 1, and the
simian rotavirus SA-11. Preparation of stocks and assay of
viruses as plaque forming units were performed as described
in Chapter 2, except that all stocks were resuspended in
antibiotic free PBS + 2% fetal calf serum. The rotavirus
SA-11 was assayed in microtiter plates using cytopathic
effect as described in Chapter 2. The 50% tissue culture
infective dose (TCID50) was determined by the method of Reed
and Muench (1938).
Digesters for Viral Studies
Aerobic digestion of sludge was performed in 10 gallon
aquaria (40 x 20 x 20 cm) containing 15 liters of sludge
Figure 3-1. Diagram of the laboratory scale aerobic
digester used in this study.
Figure 3-2. Diagram of the laboratory scale anaerobic
digester used in this study.
Gas Collection System
(Figures 3-1 and 3-2). Mixing of sludge was provided using
stirrers powered by external motors. Sludge was aerated
with humidified air introduced through air-stones in the
corners of each tank. Temperature was raised using aquarium
heaters and digesters were placed in a refrigerated
incubator for temperatures below ambient room temperature.
Anaerobic digestion was performed in 10 liter glass
carboys (after P.H. Smith, personal communication)
containing 8 liters of sludge. Temperature was controlled
by placing the digester in a heated waterbath maintained at
280C or 340C. Gas produced was collected in a 5 gallon
glass carboy and volumes produced were measured as an
indicator of digester operation.
Assay of Viruses in Digesters
Total virus was determined by mixing a 20 ml portion
of sludge with an equal volume of 6% beef extract, pH 9.0.
for one minute using a Tekmar homogenizer (Tekmar Corp.,
Cincinnati, OH) or sonicating at maximum output for 1 minute
using a Branson sonifier. The mixture was centrifuged at
14,000 x g for 10 minutes. The supernatant was neuralized
with 1M glycine pH 2.0 and diluted in PBS + 2% FCS. Diluted
and direct samples were stored at -700C until assayed.
Virus in the liquid fraction of the sludge was assayed
by centifuging the sludge at 500 x g for 10 minutes to
separate the flocs from the liquid portion. A portion of
the supernatant was asayed directly or after dilution in PBS
+ 2% FCS.
Sources of Sludge
Aerobically digested and waste activated sludge were
obtained from the Gainesville Main Street, Gainesville
Kanapaha, and University of Florida wastewater treatment
plants (see Chapter 2). Anaerobically digested sludge used
as the initial inoculum for the anaerobic digester was
obtained from the city of Tallahassee wastewater treatment
Physical and chemical analyses of sludge were
performed as described in Chapter 2.
Procedures for Studying Survival of Viruses
Virus survival was studied in 15 trials which lasted
from 4 to 10 days. Two to four digesters were operated
during each trial under different conditions as described in
Table 3-1. Digesters were set up with an initial seed of
sludge and operated 2-4 days prior to addition of virus to
achieve the desired operating conditions. Viruses were
added at the beginning of the run by inoculating the desired
concentration (105/ml) into the appropriate volume of waste
activated sludge and mixed for 1 hr prior to addition of
sludge to the digesters. To better simulate actual
operating conditions virus was added at the initial
concentration with daily feeding of sludge to obtain the
desired detention time, 15 or 40 days. In addition two
runs were performed without daily feeding of virus to obtain
a regression equation for die off.
Calculation and Statistical Analysis of Results
Virus removal was calculated as Logl0 daily change
using the following formula:
Log daily change
=LOG10 (virus recovered dayn)
(virus recovered dayn-l+ virus added dayn-1)
*All virus concentrations in PFU/ml.
These values were then used to calculate the mean daily-
change for each run and treatment (Table 3-2) and analyzed
using the Statistical Analysis System (SAS Institute
Incorporated 1982) provided by the Northeast Regional Data
Center, University of Florida.
Summary of treatments studied in laboratory scale digesters.
Dissolved Volatile ion
Temperature oxygen Total solids solids concentration
Treatment* Virus oC (mg/1) (g/1) (g/1) (M)**
Poliovirus 1 28 5.8 10.4 4.8 4 (5.4)
1 Echovirus 1 27.4 4.8 ND+ ND 4.6 (5.3)
Coxsackie B3 24.6 5.7 ND ND 1.4 (5.9)
Rotavirus SA-11 28.9 6.2 ND ND 0.7 (6.2)
2 Poliovirus 1 17.6 5.2 7.9 5.7 1 (6.0)
Poliovirus 1 5.5 5.8 19.8 5.2 1 (6.0)
3 Echovirus 1 5.0 6.0 ND ND 0.4 (6.4)
Rotavirus SA-11 3.2 7.3 ND ND 0.1 (7.0)
---------------------------- -------------------- ------------------
Poliovirus 1 27.9 0.88 8.6
Poliovirus 1 28.2 2.7 20.6
Poliovirus 1 27.1 5.1 9.1
Poliovirus 1 28 6.3 6.4
Poliovirus 1 28.7 4.4 8.9
Poliovirus 1 28.1 5.6 12.5
Echovirus 1 27.3 4.8 ND
These numbers will be referred to in subsequent text and tables.
pH in parenthesis.
-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- ----------------------------
- - - -
Results and Discussion
Effect of Sludge Source on Poliovirus Survival
Table 3-3 contains the comparison of virus removal by
sludges from different sources under conditions considered
typical for aerobic digestion. It can be seen that removal
of poliovirus was not affected by the source of sludge used
for the digester runs. Therefore all subsequent comparisons
can be made ignoring the sludge source.
Effect of Digestion Temperature on Poliovirus Survival
Table 3-4 contains the results obtained when all
digestion conditions were held constant except digestion
temperature. These results show that as digestion
temperature was decreased the removal rate of poliovirus was
decreased. Table 3-4 also shows the comparison of the
operation parameters used to monitor the digester operation.
The three temperatures were found to be significantly.
different. Total solids showed a significant increase at the
lowest temperature (treatment 3). The hydrogen ion
concentration was different for treatment 1. Figure 3-3
shows the results of a run without daily seeding of
poliovirus. This figure illustrates the difference in
removal rates between the digestion temperatures of 289C and
5C. It can be seen from the slope of the regression lines
Effect of sludge source on Poliovirus (type 1, Lsc) survival in laboratory
scale aerobic digesters.*
Dissolved Total Volatile Mean daily
Sludge Temperature oxygen solids solids change
source (oC) (mg/1) (g/1) (g/1) (log10)
U.F. campus 28A 6.3B 6.4B ND** 0.97A
Gainesville 28.7A 4.4C 8.9A 6.1A 0.71A
Gainesville 28.1B 5.6B 12.5A 4.2B 0.87A
* Means within the same column with the
different at the p=0.05 level.
same letter are not significantly
** not done.
Table 3-4. Effect of temperature on Poliovirus (type
scale aerobic digesters.*
1, Lsc) survival in laboratory
Dissolved Total Volatile Mean daily
Temperature oxygen solids solids Hydrogen ion change
(C) (mg/1) (g/l) (g/1) (10-PH)** (logl0)
28A 5.8A 10.4B 4.8A 4.2 x 10-6A 0.77A
17.6 5.2A 7.9B 5.7A 1.1 x 10-6B 0.5B
5.5C 5.8A 19.8A 5.2A 5.5 x 10-7B 0.21C
* means within the same column with the same letter are not significantly
the p=0.05 level.
** pH in parenthesis.
Figure 3-3. The effect of temperature on poliovirus
inactivation during aerobic sludge digestion.
(Least Squares derived line).
that there was a large difference between the two
temperatures, -0.03 log/day at 59C and -0.21 and -0.17
log/day at 280C. Figure 3-4 shows the difference between
log daily change at 289C and 50C. It is again easy to see
the difference in virus removal at these two temperatures,
with the lower temperature remaining close to 0 log
change/day. These results compare to those found by others
for anaerobic digestion. Eisenhardt et al. (1977) found a
1.1 log increase in virus removal when digestion temperature
was increased from 320C to 350C. Sanders et al. (1979)
studied the survival of poliovirus at 340C and 370C under
anaerobic conditions in the laboratory and found that
temperature had a significant effect on the loss of viruses
in their digesters. In addition, Ward et al. (1976) found a
decrease of virus removal with a decrease in temperature
from 280C to 40C. It should be noted that in the latter two
studies a similar effect of temperature was found in the-
absence of sludge.
Figure 3-4. Log daily change in poliovirus at 280C and 5C
during aerobic sludge digestion.
.- 0-o 5C
1 2 3 4 5 6 7 8 9 10 11 12
Effect of Dissolved Oxygen on Poliovirus Removal
The effect of dissolved oxygen is displayed in Table
3-5. It can be seen that as the DO (dissolved oxygen) was
decreased in the aerobic digesters the reduction in
poliovirus numbers was not affected significantly. In fact
the reduction was actually greatest for the aerobic
treatment with the lowest DO (0.9 mg/1). However, under
anaerobic digestion conditions the removal of poliovirus was
significantly reduced. This indicates that the removal of
poliovirus may be controlled by some process which operates
even at low DO levels. It should be noted that the lower
levels of DO are closer to those found in full scale
digesters (see Table 5-2).
It is interesting that despite the significantly
higher temperature in the anaerobic digester the virus
removal rate was significantly lower than in the aerobic
digesters. This indicates that while thermal inactivation
is important there is some function of aerobic digestion
.which appears to accelerate virus removal. A second
possibility is that the protective component found in
anaerobically digested sludge (Ward and Ashley, 1976) is not
found in aerobically digested sludge.
Figures 3-5 and 3-6 illustrate the difference in daily"
change for anaerobic versus aerobic digestion and for
Effect of dissolved oxygen on Poliovirus (type 1, Lsc) survival in
laboratory scale sludge digesters.*
Dissolved Total Volatile Mean daily
Temperature oxygen solids solids Hydrogen ion change
(C) (mg/1) (g/1) (g/1) (10-pn)** (logl0)
28B 5.8A 10.4C 4.8B 4.7 x 10-6A 0.77
32.4A 0D + 15.5 9.3A 3.8 x 10-7B 0.33A
27.9B 0.88C 8.6C 2.6C 1.4 x 10-7B 1.03B
28.28 2.7B 20.6A D++ 2.3 x 10-6AB 0.91B
* Means within the same column with
the p=0.05 level
** pH in parenthesis.
+ anerobic digestion.
+ Not done.
the same letter are not significantly different at
Figure 3-5. Comparison of daily change in poliovirus during
aerobic and anaerobic sludge digestion.
-5.0 mg/L 02
1 2 3 4 5 6 7 8 9 10
Figure 3-6. Daily change in poliovirus during aerobic
sludge digestion at 1.0 and 5.0 ppm dissolved
0--0 5.0 mg/l 02
.----6 1.0 mg/l 02
-3.0 1 1 1 --1-
1 2 3 4 5 6 7 8 9 10
aerobic digestion at different DO levels. These figures
show the same trends described in Table 3-5. The daily
change for anaerobic digestion was lower than that of
aerobic digestion. Secondly, dissolved oxygen does not
affect virus removal significantly during aerobic digestion
at concentrations between Ippm and 5ppm.
Effect of Detention Time on Poliovirus Survival
Table 3-6 contains the comparison of runs with similar
operating conditions excluding detention time. Two
detention times were compared, 16 days and 40 days.
Although the 40 day detention time resulted in a greater
virus removal no significant difference was found between
the two detention times. Sanders et al. (1979) found that
detention time played a decreasing role as digestion
temperature was increased for anaerobic digestion. They
found that increasing the detention time by 5 days at 37C
resulted in a 1 percent loss of recoverable infectivity.
However, at 340C, increasing the detention time 5 days
resulted in an 11 percent loss of recoverable infectivity.
It is possible that a similar condition exists for aerobic
Effect of detention time on Poliovirus (type 1, Lsc) survival in laboratory
Detention Dissolved Total Volatile Mean daily
time Temperature oxygen solids solids change
(days) (C) (mg/1) (g/1) (g/1) (log10)
16 28A 5.8A 10.4A 4.8A 0.77A
40 27.1B 5.1B 9.1A 5.9A 0.98A
Means within the same column with the same letter are not significantly different at
digestion and the digestion temperature used (280C) was that
temperature where detention time begins to decrease in
Correlation of Operation Parameters withVirus Removal
Table 3-7 contains correlation coefficients for the
various operating parameters. Examining the correlation
coefficients for virus removal versus the various
parameters, note that DO was significantly correlated with
virus removal. Since virus removal is expressed as logl0
daily change the more negative the number the greater the
removal, therefore DO was negatively correlated with virus
removal. Notice that DO was positively correlated with
volatile solids and virus removal was negatively correlated
with volatile solids. This could indicate that volatile
solids reduction is linked to virus removal and as dissolved
oxygen was increased volatile solids also increased due to
an increase in biomass. This could also indicate that virus
removal is controlled by some reaction which occurs more
efficiently at lower oxygen levels possibly due to a slower
removal of some virucidal agent.
Table 3-8 contains the correlation coefficients for
the anaerobic digester runs. Although not highly
significant, virus removal was positively correlated with
Correlation matrix for aerobic digesters.
Dissolved Total Volatile Log daily
oxygen Temperature solids solids change
Log daily 0.21 -0.39 0.29 0.37
change (0.006)* (0.0001) (0.04) (0.029)
Total -0.07 -0.45 --- 0.09
Solids (0.63) (0.0005) (0.58)
Volatile 0.38 -0.22 0.09
solids (0.024) (0.185) (0.578)
Hydrogen ion 0.15 0.26 -0.048 0.052 0.118
concentration (0.057) (0.0005) (0.73) (0.76) (0.13)
values in parenthesis indicate probability level for correlation coefficient.
Correlation matrix for anaerobic digesters.
Total Volatile Log daily
Temperature solids solids change
Log daily -0.24 -0.398 -0.699
change (0.19)* (0.29) (0.19)
Total -0.85 --- 0.89
solids (0.003) (0.04)
Hydrogen ion -0.39 0.69 0.299 0.18
concentration (0.03) (0.06) (0.62) (0.33)
Values in parenthesis are probability level for correlation coefficient.
total and volatile solids, in other words as these
parameters increase virus survival decreases. This is
similar to what Sanders et al. (1979) found. However, it is
also contrary to what is reported in Table 3-7 for aerobic
digestion. This finding as well as the results with
temperature indicates different removal mechanisms for
aerobic and anaerobic digestion.
Effect of Virus Type on Removal Rate
Table 3-9 contains the comparison of removal rates for
several virus types by the treatments described in Table 3-
2. No significant difference was found for any of the
viruses regardless of treatment. Several other workers
(Bertucci et al., 1975; Bertucci et al., 1977) have found
that viruses belonged to distinct groups according to
removal rate. None of the viruses tested here demonstrated
that trend, and although poliovirus was removed at a greater
rate for treatment 1 this was not the case for the other
treatments. It is also important that the simian rotavirus
SA-11 was found to behave similarly to the three
Comparison of virus survival during aerobic
digestion of wastewater sludges at various.
Mean daily Dissolved
change Temperature oxygen
Treatment* Virus (logl0 ** (C) (mg/1)
Poliovirus -0.77A 28.9 6.2
(type 1, Lsc)
Echovirus 1 -0.5A 24.6 5.7
Coxsackievirus -0.46A 27.4 4.8
Rotavirus -0.43A 28.0 5.8
Poliovirus -0.21B 5.5 5.8
(type 1, Lsc)
3 Echovirus 1 -0.18B 5.0 6.0
Rotavirus -0.44B 3.3 7.3
Poliovirus -0.33C 32.4 +
(type 1, Lsc)
4 Echovirus 1 -0.53C 31.4
Rotavirus -0.52C 34.0
* Treatments described in Table 3-2.
** Means with the same letter for each treatment are not
significantly different at the p=0.05 level.
Distribution of Viruses in Sludge Components
Table 3-10 contains the percent virus in the liquid
fraction of sludge. It can be seen that more than 90
percent of the virus in the sludges, for the various
treatments (see Table 3-2), are solids-associated. This is
interesting especially when considering the data that
indicate that virus removal is linked to solids removal and
production. It is also important in relation to development
of methodology for recovery of indigenous viruses from
The removal of viruses in aerobic scale digesters was
found to be significantly greater than the removal in
anaerobic digesters. The sludge source and detention time
were not found to affect the removal rate of viruses.
Although a significant difference was found between aerobic
and anaerobic digestion, there was no significant difference
between virus removal rates at dissolved oxygen levels
ranging from 0.9 to 5.8 mg/l. However, dissolved oxygen
level was negatively correlated with virus removal. These
findings indicate that the processes linked to virus removal
Table 3-10. Percent virus in sludge supernatant
for various treatments used in
laboratory scale digesters.
in the sludge
(mean + s.d.)
1 1.02 + 2.73
2 0.22 + 0.377
3 1.32 + 1.72
4 1.39 + 1.7
5 0.02 + 0.41
* Treatments described in Table 3-2.
under aerobic digestion conditions are somehow controlled by
DO. The single most important factor affecting virus
removal was temperature. Virus removal was also negatively
correlated with volatile solids indicating that the
processes controlling volatile solids reduction are also
involved in virus removal. The virus type was not found to
affect the removal rate of virus during aerobic or anaerobic
DEVELOPMENT OF METHODOLOGY FOR RECOVERY OF
VIRUSES FROM AEROBIC SLUDGES
The quantitative recovery of virus from sludges has
been one problem delaying the effective study of virus fate
during wastewater sludge treatment. Many workers have
attempted to develop and use such a method (Table 4-1).
However, most of these techniques have been developed for
use without considering sludge type or comparing the
recovery of seeded with indigenous viruses. If useful data
are to be obtained the above mentioned factors and the
criteria in Table 4-2 must be considered.
Viruses found in sludges have been shown to be
primarily solids-associated (Glass et al., 1978; Hurst et
al., 1978; Lund, 1971; Ward and Ashley, 1976; Wellings et
al., 1976) and consequently any elution procedure must be
able to recover these solids-associated viruses. The
Table 4-1 Summary of published methods for the recovery of viruses from wastewater sludges.
Elution Concentration Sludge
Method Method Virus Type Reference
(0.05M),pH 11.5 +
or freon extraction
filtration of floc
Poliovirus type 1
Mixed liquor and
Glass et al. (1978)
Hurst et al. (1978)
Wellings et al. (1976)
3% casein None
3% fetal calf serum
wrist action shaking
SSattar and Westwood (1976)
Acid precipitation None
pH 7.0, Alum
and Tris pH 9.0
elution with 10%
beef-extract pH 7.0
Nielsen and Lydholm
2% FCS pH 9.5 +
in McIlvaine buffer
pH 7.0 + magnetic
Poliovirus type 1
Turk et al. (1980)
Moore et al. (1979)
Berg and Berman
Table 4-1 continued
Elution Concentration Sludge
Method Method Virus Type Reference
saline pH 9.8
citric acid buffer
pH 7.0 + magnetic
Fetal calf serum
Vasl and Kott
Kabrick et al.
Sattar and Westwood
0.1% SDS + 0.05M
glycine pH 7.5
(3 elution steps)
3% beef-extract Poliovirus type 1 Anaerobically
organic flocculation digested
Abid et al. (1978)
0.4M urea + 0.1M
lysine pH 9.0 +
Poliovirus type 1
Echovirus type 1
Farrah et al.
Table 4-2. The criteria used to evaluate methods for
recovery of viruses from wastewater sludges.
1. Simple and inexpensive to perform.
2. Achieve a high virus recovery for all sludge types.
3. Include a concentration step to minimize the necessary
plating volume, consequently reducing the number of
tissue cultures required.
4. Free from toxicity and contamination problems.
predominant theory involving virus adsorption to surfaces is
that electrostatic forces are involved (Bitton 1975).
Recently Farrah and coworkers have reported that hydrophobic
interactions may also be involved in viral adsorption to
membrane filters and possibly other surfaces (Farrah et al.,
1981C; Farrah, 1982; Shields and Farrah, 1983).
Consequently, elution of viruses has been attempted
utilizing procedures which minimize electrostatic
interactions. Such procedures involve raising the pH above
the isoelectric point of viral proteins (Farrah et al.,
1981B; Hurst et al., 1978; Nielsen and Lydholm, 1980;
Subrahmanyan, 1977; Vasl and Kott, 1981; Wellings et al.,
1976), lowering the ionic strength of the solution (Turk et
al., 1980), and using amino acid or protein solutions to
compete with viruses for available adsorption sites (Abid
et al., 1978; Berg and Berman, 1980; Farrah et al., 1981B;
Glass et al., 1978; Hurst et al., 1978; Kabrick et al.,
1979; Nielson and Lydholm, 1980; Sattar and Westwood, 1976;-
Sattar and Westwood, 1979; Subrahmanyan, 1977; Wellings et
al., 1976). Detergents such as sodium dodecyl sulfate have
also been used (Abid et al., (1978).
Mechanical disruption of sludge flocs has been
included in an attempt to free embedded viruses. The methods
include blending (Glass et al., 1978; Subrahmanyan, 1977;
Turk et al., 1980), sonication (Glass et al., 1978;
Wellings et al., 1976), shaking (Sattar and Westwood, 1976;
Sattar and Westwood, 1979), and vigorous mixing on a
magnetic stirrer (Berg and Berman, 1980; Farrah et al.,
1981B; Hurst et al., 1978; Kabrick et al., 1979; Wellings et
Sludges may often contain very low concentrations of
viruses. This poses a problem time wise and economically,
since large volumes of sludge eluate must be assayed to
detect low numbers of viruses. This would require the use
of a significant number of tissue cultures resulting in high
material cost and technician time required for these assays.
To minimize these costs it is advantageous to concentrate
sludge eluates to smaller volumes.
Several procedures have been used to concentrate
viruses from sludge eluates (Table 4-1). The most common
concentration procedure is the organic flocculation
procedure ( Abid et al., 1978; Farrah et al., 1981B;
Glass et al., 1978; Hurst et al., 1978; Kabrick et al.,
1979). This method was developed for use with beef-extract
by Katzenelson et al., (1976) and has been used for other
protein solutions such as casein (Bitton et al., 1979) and
glycine (Hurst et al., 1978). Organic flocculation is
based on the principle that adjusting a protein solution to
the isoelectric point of the protein results in a net
neutral charge on the protein molecules resulting in the
formation of flocs. Virus particles are adsorbed by these
flocs, which are easily removed by filtration or
centrifugation and subsequently dissolved in a smaller
volume of buffer. Chemical flocculation can also be used
with compounds such as aluminum hydroxide which forms flocs
at neutral pH (Farrah et al., 1981B; Hurst et al., 1978).
Low pH adsorption of eluted viruses to bentonite clay
(Turk et al., 1980) and filters (Hurst et al., 1978)
followed by subsequent elution in smaller volumes has also
been utilized. Concentration by polyethylene glycol
hydroextraction (Wellings et al., 1976) involves the
reduction in liquid volume by placing dialysis bags
containing the sludge eluate in polyethylene glycol until
the eluate is reduced to the desired volume.
The purpose of this chapter is to evaluate methodology
for the detection and quantification of viruses in sludges.
A primary goal was to develop a procedure for use with
aerobic sludges. The selection procedure was performed by
evaluating published methodology and methods developed in
this lab. A two step evaluation was employed as described
in Table 4-3.
Several of the methods listed in Table 4-1 are not
appropriate for use with aerobic sludges as they do not
Table 4-3. Protocol for evaluating candidate methods to
recover viruses from aerobically digested
Step-i Evaluate published methods and in-house methods
using several types of virus-seeded sludges.
Step-2 Evaluate top candidate methods for recovery of
indigenous enteroviruses from several sludge
* Part of this evaluation process was performed while
testing methods for inclusion as an ASTM standard
include concentration steps (Berg and Berman, 1980; Lund,
1973; Nielsen and Lydholm, 1980; Sattar and Westwood, 1976;
Sattar and Westwood, 1979; Subrahmanyan, 1977; Vasl and
Kott, 1981). This leaves the methods of Farrah et al.
(1981B), Glass et al. (1978), Hurst et al. (1978), Kabrick
et al. (1979), Turk et al. (1980), and Wellings et al.
(1976) as possible candidates along with several in-house
methods to be evaluated for use with aerobic sludges. It
should be noted that the methods of Glass et al. (1978),
Kabrick et al. (1979), Turk-et al. (1980), and Wellings et
al. (1976) or some modification were simultaneously being
evaluated for possible inclusion as an ASTM standard method.
Materials and Methods
Viruses and Viral Assays
Poliovirus type 1 (Lsc) and Poliovirus type 1 (Sabin)
were used to evaluate the methods in laboratory experiments:
Poliovirus type 1 (Sabin) was used to evaluate the method of
Hurst et al. (1978), and all other methods were evaluated
using Poliovirus' type 1 (Lsc). Preparation of stocks and
assay of viruses as plaque forming units were performed as
described in Chapter 2.
Indigenous viruses were detected using BGM cells by
cytopathic effect and plaque assay as described in Chapter
2. The 50% tissue culture infective dose (TCID50) was
determined by the method of Reed and Muench (1938).
The chemicals used and their sources are glycine,
ethylenediaminetetraacetic acid (EDTA), urea, sodium
trichloroacetic acid (TCA), and lysine (Sigma Chemical
Company, St. Louis, Mo.), isoelectric casein and purified
casein (Difco Laboratories, Detroit, Mich.), beef-extract
(Inolex Corp., Glenwood, Ill.), Antifoam B (J. T. Baker
Chemical Company, Phillipsburg, N. J.), bentonite and
chloroform (Fisher Scientific Company, Pittsburg, Pa.).
Sludges used in seeded studies are described in Table
2-1. All samples were used for seeded studies within one
week of collection and were stored at 4QC until used.
Before use sludge samples were brought to room temperature.
Sludges used in indigenous and ASTM Studies are described in
Methods for Virus Detection
The-methods evaluated are those described in Table 4-1.
Stock virus in PBS containing 2% FCS was added directly to
100, 250, 500, or 1000 ml of sludge while stirring on a
magnetic stirplate. Magnetic stirring was continued for 60
minutes. At the end of the contact period a sample of the
sludge was diluted without removal of solids and used to
calculate the initial virus concentration. This value was
compared to the value expected from the stock titer.
Pancorbo (1982) has shown that this procedure does indeed
correspond to the virus load expected from the stock titer.
The sludge was then processed according to the protocol of
the method being evaluated.
Method-1: Glycine high pH (Hurst et al., 1978). The
method used was a modification of the protocol reported by
Hurst et al. (1978) and was performed as follows:. the sludge
sample was centrifuged at 4,080 x g for 10 minutes. The
supernatant and sludge pellet were both processed for virus
The pellet was mixed with five volumes -of 0.05M'
glycine buffer, pH 11.5. The pH of the mixture was adjusted
to 10.5-11.0 by the addition of 1M glycine buffer, pH 11.5.
The sample was mixed for 30 seconds on a magnetic stirplate
and centrifuged at 1,456 x g for 5 minutes. The supernatant
was then concentrated by organic flocculation, using the
organic from the sludge present in the pellet eluate.
The supernatant was adjusted to pH 3.5 by the addition
of 0.05M glycine pH 2.0. The flocs produced were removed by
centrifugation at 1,456 x g for 20 minutes. The resulting
pellet was eluted with 5 volumes of PBS plus 2% FCS at pH
9.0, centrifuged at 3,020 x g for 5 minutes, neutralized and
assayed. The organic flocculation supernatant was passed
through a 3.0, 0.45, 0.25 jm Filterite filter series.
(Filterite Corp., Timonium, Md.). Adsorbed virus was eluted
with 7 ml of PBS plus 2% FCS at pH 9.0, additional 3 ml
fractions were passed through the filters until the filtrate
was pH 9.0. The filtrate fractions were combined,
neutralized, and assayed.
The supernatant produced in the first centrifugation
to fractionate the sludge was neutralized and assayed for
virus without concentration, in most cases. When
concentrated it was treated in the same manner as the
organic flocculation supernatant.
Method 2: Beef-extract-hydroextraction (Wellings et
al., 1976). The procedure used was from a protocol supplied
by Dr. F. M. Wellings to ASTM task force D-19:24:04:04/05.
The sludge was supplemented with sufficient, cold, 30%
beef-extract in distilled water to obtain a final
concentration of 3%. The sample was mixed in a Waring
blender while slowly increasing and decreasing the speed
using a rheostat. The sample was defoamed by mixing on a
magnetic stirplate for 10-minutes. The pH was adjusted to
9.0 with 1N NaOH. The sample was subsequently sonicated for
15 minutes at maximum output in a rosette cooling cell
submerged in an ice bath. The sonicated sample was
centrifuged at 1200 x g for 30 minutes. The supernatant was
removed and concentrated by polyethylene glycol
hydroextraction. The concentrated sample was washed out of
the dialysis bag with 2-5 ml of PBS pH 7.2, with gentle
kneading. The sample was subsequently filtered through a
25 mm, 0.45 pm Millipore filter (Millipore Corp, Bedford,
Mass.), that had been pretreated with FCS, and assayed for
Method 3: Distilled water-bentonite adsorption (Moore
et al., 1979). This method was performed according to a
protocol supplied by Ms. B. E. Moore to ASTM task force D-
The sludge sample was centrifuged for 10 minutes at
4,080 x g and the supernatant and pellet were processed for
virus assay as follows:
Bentonite clay and CaC12 were added to the supernatant
to a final concentration of 100 ppm and 0.01M, respectively.
The pH was adjusted to 6.0 and the sample was mixed for 30
minutes. Following mixing the sample was centrifuged for 20
minutes at 4,080 x g. The resulting pellet was mixed with
an equal volume of tryptose phosphate broth. After shaking
for 15 minutes the sample was centrifuged at 12,100 x g for
15 minutes and the resulting supernatant was assayed for
The sludge solids were mixed with an equal volume of
sterile distilled water, homogenized in a Waring blender for
3 minutes at low speed. The sample was centrifuged for 20
minutes at 4,080 x g, and the resulting supernatant was
treated as described above for the sludge supernatant.
Method 4: Beef-extract-organic flocculation (Glass et
al., 1978). The protocol for this procedure was as
published by Glass et al. (1978) and supplied to ASTM task
group D- 19:24:04:04/05.
An 800 ml sludge sample was placed in a sterile
blender jar, 19.2 g of beef-extract powder and 0.4 ml
antifoam B were added and the sample was blended for 1
minute at low speed followed by 2 minutes at high speed.
The suspended sludge was transferred to a sterile beaker,
adjusted to pH 9.0 with 2N NaOH and mixed for 15 minutes on
a magnetic stirplate. The sludge was transferred to sterile
centrifuge bottles in an ice bath and sonicated for 2
minutes at maximum output. The sample was then centrifuged
at 10,000 x g for 30 minutes. The supernatant was
concentrated by organic flocculation using 2N HCL to lower
the pH to 3.5. The acidified supernatant was mixed for 30
minutes on a magnetic stirplate and subsequently centrifuged
at 10,000 x g for 30 minutes. The resulting pellet was
resuspended in a single 10 ml aliquot of 0.15M sodium
phosphate, pH 9.0, and the pH was adjusted to 7.5 when
necessary. For the detection of indigenous viruses the
final sample was mixed with 10 ml of-dithizone working stock
(see appendix) for 1 minute on a vortex. The dithizone-
sample mixture was centrifuged at 10,000 x g for 30 minutes,
the upper aqueous phase was placed in a sterile test tube,
0.05 ml of sterile 1M CaC12 was added and the sample was
bubbled with air for 10 minutes. To this final sample 0.8
ml of a 10,000 unit/ml penicillin stock, 0.3 ml of 25,000
pg/ml steptomycin stock, and 0.15 ml of 500 pg/ml of
fungizone stock were added.
Method 5: Urea-Lysine Method (Farrah et al., 1981B).
Sludge samples were centrifuged as 4,080 x g for 5 minutes.
The supernatant and pellet were treated separately.
The supernatant was adjusted to 0.003M aluminum
chloride, adjusted to pH 7.0 by the addition of 1M sodium
carbonate, and mixed for 5 minutes. The pellet was mixed
with 2-5 ml of 3% beef-extract and 0.1M EDTA pH 9.0. After
mixing by magnetic stirring 10-15 minutes, the sample was
centrifuged at 16,300 x g for 15 minutes. The supernatant
was neutralized and dialyzed in PBS pH 7.2 plus antibiotics
overnight at 4C. The dialyzed sample was adjusted to pH
3.5 by addition of 1M glycine pH 2.0. The floc was
collected by centrifugation at 3,090 x g for 5 minutes and
dissolved in 1-2 ml of 0.15M sodium phosphate.
The sludge pellet was mixed with 5 volumes of freshly
prepared 4M urea plus 0.05M lysine, pH 9.0, for 5 minutes.
The pH of the sludge-urea mixture was adjusted to 9.0 with
1M lysine pH 11.5. The sample was centifuged at 4,080 x g
for 5 minutes, the supernatant was neutralized by the
addition of 1M lysine pH 2.0. The sample was then adjusted
to 0.005M aluminum chloride and the pH readjusted to 7.0 by
the addition of 1M sodium carbonate and mixed 5-10 minutes.
The floc was collected by centrifugation at 4,080 x g for 5
minutes and treated as described in the previous paragraph.
Method 6: Casein-Glycine Method. The casein-organic
flocculation procedure is a modification of the procedure
used by Bitton et al. (1979) for concentration of
enteroviruses from seawater.
The final modified procedure was performed as
described in the subsequent text. Sufficient 5% purified
casein in 2.5M glycine pH 9.0 to achieve a final
concentration of 1% casein and 0.5M glycine was added to
the sludge. The pH was adjusted to 9.0 with 1M glycine pH
11. Antifoam B was added and the mixture was blended for 2
minutes at maximum speed, the pH was readjusted to 9.0 and
the sample was mixed for 10 minutes on a magnetic stirplate.
The mixed sample was centrifuged for 10 minutes at 16,300 x
g. The supernatant was decanted, adjusted to pH 7.0, and
concentrated by either organic flocculation or polyethylene
Hydroextraction was performed as previously described
for the method of Wellings et al. (1976). Organic
flocculation was performed by adjustment of the pH of the
supernatant to 4.5 with 1M glycine pH 2.0 and then mixed for
10 minutes. The floc produced was collected by centrifuging
the sample at 16,300 x g for 10 minutes. The floc was
resuspended in 2-5 ml of 0.15M sodium phosphate pH 9.0 and
subsequently centrifuged for 10 minutes at 12,000 x g. The
sample was decontaminated by addition of 0.1 volume of
chloroform, the sample shaken for 15 minutes, and the
chloroform was removed by bubbling filtered air vigorously
for 30 minutes, past the the point where no chloroform odor
was detected. Penicillin and steptomycin at cell
concentrations (see appendix) were added and the sample was
assayed for virus.
Method 7: TCA-Lysine Method. The sludge sample was
adjusted to pH 3.5 by the addition of 1M glycine pH 2.0 and
mixed for 30 minutes on a magnetic stirplate. The sample
was subsequently centrifuged for 20 minutes at 4,080 x g.
The supernatant was discarded and the pellet was mixed with
5 volumes of 1M TCA plus 0.1M lysine pH 9.0, and mixed 15
minutes on a magnetic stirplate. The sample was
centrifuged 5 minutes at 4,080 x g. The supernatant was
collected and neutralized with 1M glycine pH 2.0. The
neutralized supernatant was treated as described for the
Results and Discussion
Evaluation of Seven Methods for
Recovery of Poliovirus 1 from Sludges
Method-1: Glycine high pH (Hurst et al., 1978). Hurst
et al. (1978) evaluated their procedure on mixed liquor and
found that viruses added to activated sludge could be
recovered at efficiencies of 80-84% for poliovirus (type 1,
Chat), 68% for echovirus (type 7), and 75% for
coxsackievirus (type B3). They then applied their method to-
the study of virus in several sludge types.
Table 4-4 shows the results of work done in this lab
evaluating the Hurst et al. method for several sludge types.
Poliovirus 1 recoveries from the two mixed liquor samples
were similar to those of Hurst et al. (1978), 72% and 80%
respectively. Also of interest is the recovery of
poliovirus from aerobic sludges, these results show a
Recovery of Poliovirus (type 1, Lsc) from sludges using the method
of Hurst et al. (1978).
Volume Total virus Mean % virus
processed Concentration added % virus recovered by
Sludge* (ml) Factor** (PFU X 106) recovered sludge type
UML 100 --+ 8.4 84.6
GML 1000 30 1.3 60.3 72.3A
GW 1000 5 7.4 8.3
UDA 500 -- 6.5 18.9
UDA 500 -- 6.5 11.9
UDA 500 -20.0 8.0
UDA 500 -- 20.0 5.9 14.5 + 7B
UDA 1000 17 9.6 15.3
GDA 90 1000 17 13.0 14.1
PDA 1000 2 4.1 26.9
PDAN 100 -- 6.0 59.9
PDAN 1000 -- 5.5 63.9 60.2 + 3.6A
LAG+ 1000 4 1.8 56.7