United States
Environmental Protection
Agency
Environmental Monitoring and Suppqrt
Laboratory
Cincinnati OH 45268
Research and Development
EPA-600/S4-83-012 May 1983
Project Summary
Quantitation of Viruses in Waste
and Other Waters
James E. Smith
During treatment of domestic sew-
age by the activated sludge process,
large numbers of viruses are inactivated,
principally by adsorption to the micrc-
bialfloc. The numbers of viruses which
are removed by adsorption vary over a
wide range due to largely unknown
factors. This study describes the inter-
actions between the virions and ex-
opolysaccharide surfaces of gram neg-
ative, u rear/tic floe bacteria and de-
monstrates adsorption of virus particles
by extracellular polyhydroxybutyrate
granules produced by Zoogloea ram-
igera, a common sludge floe organism.
Zoogloea ramigera was used as a
model system to study the kinetics of
virus removal by adsorption. By varying
carbohydrate levels, large amounts of
extracellular polysaccharides or extra-
cellular polyhydroxybutyrate granules
were obtained; both materials avidly
absorbed 125 I-labeled enteroviruses
and some phages in a nonspecific fash-
ion. Adsorbed viruses could be removed
by alkaline extraction and/or displace-
ment with organic competitors.
Factors which affect the virus-ex-
opolysaccharide interactions include
the species of organisms, presence of
heavy metals and multivalent cations,
bulking conditions of the sludge treat-
ment, pH, and humic acid or other
organic compounds. Some virions are
released from these complexes as the
sludge floes disintegrate during vari-
ous phases of sewage treatment. The
presence of bacterial polysaccharide
particles can be detected on the virus
coats.
In the laboratory viruses can be trans-
ferred to protozoa which graze on the
floes in aerobic treatment processes
and on the suspended solids in effluent
Purified entero-, reo-, adeno-, and SV40
viruses labeled with 125I by lactoper-
oxidase or chlorogrycoluril methods
were adsorbed to floes of ultraviolet-
irradiated bacteria and fed to ciliates.
The zoomicrobes were isolated and
their virus content was determined as
125I equivalents or plaque forming units.
Appreciable quantities of viruses were
acquired by several ciliates in axenic
cultures and by mixed ciliates which
were obtained by flotation from primary
sludge. The 125l-virus counts in the
ciliates were inversely proportional to
the reduced number of bacteria. Re-
tention of intact virions was determined
by using viruses labeled with3 H-uridine
and 125I. Retention times varied with
different feeding regimes. Protozoa
excreted 90 percent of the 1251 in 2 to
72 hours and small numbers of viable
poliovirus 1 were excreted. Attempts
to transfer 125l-viruses from ciliate to
ciliate or to cysts failed.
This Project Summary was developed
by EPA's Environmental Monitoring
and Support Laboratory, Cincinnati,
Ohio, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).
Introduction
Microbiologists have demonstrated rou-
tinely the presence of enteroviruses in
fecal contaminated water, sewage sludge,
and treated effluent The rate of these
viruses' natural die-off in water and sew-
age has been determined, frequently with
a comment aside that either the initial
reduction in titer or even the entire loss
could be ascribed to adsorption of the
virions by colloidal material. Although
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repeated attempts have been made to
correlate both the type and quantity of the
colloidal solids in sewage with the amount
of virus infective units lost after seeding,
the overall picture of what happens phys-
ically to the virus particles is not clear. The
enteroviruses and their components are
potentially long-lived. A large proportion
of the viruses which survive the initial
reduction at the treatment plant and then
reach the outfall will exhibit half-lives
measured in days 01 weeks instead of a
few hours.
There appear to be two methods of virus
adsorption which produce this viral pre-
sence. Reversible adsorption occurs when
viruses are adsorbed to clays, inorganic
precipitates and vegetable fiber. It occurs
very quickly after virus is added to the
water and responds to manipulation of pH
and ionic strength. Irreversible adsorption
is apparently a biological process with a
rather long lag time, has a pronounced
oxygen demand, is not clearly related to
any particular bacterial or algal species
which have been studied. Its response to
pH, solvents, and ionic strength is unpre-
dictable and not very pronounced. There
are no definite indications as to whether
this virus loss is due to irreversible adsorp-
tion or to some other factor(s). It is
striking, however, that these losses cor-
respond very well to the characteristics of
the ciliate protozoan populations in waste-
water. These populations build up ex-
ponentially during flock formation, show
seasonal variations which might explain
unusual peaks of virus occurrence, have
slower generation times than bacteria, and
maintain feeding activity which is very
sensitive to changes in temperature and
oxygen levels.
Sludge solids also contribute to the
presence of virus in fecal contaminated
water, sewage sludge, and treated effluent
Sludge solids are generated by methods
which are largely nondegradative to virions
and which effectively concentrate dilute
viruses. Consequently the types of virus
pathogens and their virus content must be
considered in the disposal of the sludges.
There also exists the possibility that after
activated sludge has concentrated viruses
by adsorption, it may release them contin-
uously into the effluent If the sludge
becomes overloaded with organic solutes
that compete for binding spots, it may
provide bursts of free virus with high
titers.
Thus, the overall objectives of the follow-
ing study have been three-fold: to deter-
mine more precisely those elements in
sludge solids which remove or inactivate
virions; to improve the technology for
recovering and assaying adsorbed, infec-
tious viruses; and to devise techniques for
controlling and improving the removal of
viruses by precipitable sludge solids or
other methods related to the recovery of
wastewaters.
Results
Centrif ugation profiles of125 l-poliovirus
seeded into anaerobic sludge samples
showed that the virus was largely concen-
trated in the supra-colloidal fraction of the
Rickert- Hunter classification (Table 1). This
fraction contains individual bacterial cells
and protozoa.
The research conducted makes clear
that some portion of the microbial popula-
tion in activated sludge can produce ex-
ocellular polymers (glycocalyx) with a high
avidity for unenveloped viruses. Experi-
mental evidence further asserts that even
when adsorbed to fecal solids and other
colloids, viruses often are infectious and
should be assayed when the sedimentable
solids of water supplies are examined for
microbial pathogens. Adsorption may
even increase infectivity by concentrating
multiple virions on particles which can be
retained at sensitive sites in the host
Representative viruses from all the major
unenveloped viruses adhere to bacterial
floes from activated sludge. The sludge
bacteria and yeast can be divided into two
groups based on their ability to adhere 10
polystyrene plates or not. All adherent
bacteria adsorb poliovirustype 1 and other
viruses to the surface of colonies and
individual cells in the presence of Al3"1" and
Mg2+; nonadhering bacteria frequently do
not adsorb viruses or do so less efficiently.
Sludge solids were fractionated by dif-
ferential centrifugation and sucrose gradient
density centrifugation. When 125l-labeled
PV-1 was mixed with the solids before
centrifugation, most of the label was as-
sociated with either fractions containing
broken bacterial fragments and single cells
or with fractions containing large clumps
and filaments, paper debris, vegetable
fragments, and so on. The largest number
of counts were associated with fragments
1 //. diameter.
Polyhydroxybutyrate(PHB) granules from
Zoogloea ramigera are avid virus adsor-
bents. Large quantities of PHB granules
occur as a result of lysis induced by the
activated sludge process. Treatment of
the native granules with proteinases mark-
edly reduced adsorption; treatment of ad-
sorbed viruses under nondegradative con-
ditions caused the release of viruses. H
appears that the adsorption sites are lo-
cated on the proteinaceous PHB granule
membrane. The extent to which cations
are involved is unknown at this time. *
Exocellular polymers were extracted from
four strains of Zoogloea ramigera and one
Klebsiella pneumoniae type 3 and puri-
fied. The purified exopolysaccharides re-
acted very strongly with 125l-poliovirus
type 1 and either precipitated the virions
or neutralized them.
The adsorption of viruses by Zoogloea
ramigera and its cell exopolysaccharides
was mediated by the addition of A13+ and
Mg2+. Ca2+ cannot be substituted. These
large ions are involved in a complex inter-
action of the viruses and the cell surface.
The data suggest that the first interaction
of the virion and the bacterial surface is
electrostatic and results in a destabilizatior
of the bacterial surface. This is sometimes
Table 1. Comparison of Virus-Containing Sucrose Gradient Profiles of Four Digested Sludg
Samples, Metropolitan Syracuse Treatment Plant.
Fraction From
Differential
Centrifugation*
Soluble
Colloidal
Supra- colloidal
Settleable
Recovered Radioactivity** in Seeded, 5-Day
Anaerobic Sludge Samples
Sample 1
702
778
4831
1314
Sample 2
1214
2849
6084
1154
Sample 3
693
1035
4212
1742
Sample 4
1298
2152
4958
672
* Soluble: resists pelleting at 62,500 x g, 15 min
Colloidal: pellets at 62,500 x g, 15 min
Supra-colloidal: pellets at 4,800 x g, 30 min
Settleable: pellets at 1 x g, hr
** Disintegrations per min (125f) corrected for background
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represented as a"reversible sorption" and
is sensitive to EDTA interference or strip-
ping. At the end of 60 to 1 80 minutes the
virus particles can no longer be stripped
with EDTA and/or pH 1 1 glycine buffer.
The reversible sorption with the cells was
analogous to the reversible sorption of
viruses which has been observed with
membrane filters in the presence of alumi-
num ion. The secondary binding is broken
only with 4 percent beef extract, pH 9,
suggesting that metal hydroxides are being
hydrolyzed and specific superficial "do-
mams" of virus protein coat substructures
no longer mterdigitate with exopolysaccha-
ndes or glycoproteins.
Ciliated protozoa have been identified as
a major factor in the control and removal of
viruses from wastewater. They are the
dominant protozoa present in activated
sludge and their numbers are associated
with the quality of the effluent. As the
principal grazing population, they con-
sume much of the adsorbed materials in
sludge, including viruses. The transfer of
virus to cultured protozoans as well as to
wild ciliates has been demonstrated in the
laboratory when ciliated protozoans graze
on sludge floes with adsorbed virions. The
virus particles are internalized and shed
over a 12-to 48-hour period by ciliates.
Protozoa only acquire viruses which are
adsorbed to bacteria. There is no indication
that ingested viruses multiply in the ciliate
cells during this period.
Virions are released into the effluent as
the floe is comminuted by violent aeration
in activated sludge treatment The viruses,
still adsorbed to small pieces of capsular
polysacchande, are washed out in the
overflow. Thus, under bulking conditions
more viruses are to be expected in the
effluent. The virus particles released in
this manner from the floe exhibit altered
physical and biological characteristics:
they may occur singly or in clusters; their
surface is antigenically altered; their af-
finity for surfaces of rocks, water plants,
algae and debris is greatly increased; and
they may be protected to some degree
against drying and subsequent inactivation.
A reduction in suspended solids by settling
may reduce the effluent viral count but will
not eliminate single virions and small
clusters which are shed by ciliates or
released by dissolution of the floe.
Conclusions and
Recommendations
Three of the major factors that control
the distribution and survival of viruses in
sewage treatment are: (1) the exopoly-
sacchandes of floe-forming bacteria which
synthesize an abundant glycocalyx capable
of removing more than 90 percent of the
viruses by adsorption; (2) the protein
receptors and specifically charged groups
on the surface of the virus nucleocapsid
which react with the exopolysaccharides;
and (3) the virophagy and excretion of
viruses by protozoans grazing on bacterial
floes which have virions adsorbed to their
exopolysaccharide coats.
A quantitative screening method has
been devised to select organisms from
sludge floes which produce heavy glycocalyx
and adhere to polystyrene surfaces. Thus,
subcultures can be chosen which have a
high avidity for viruses. 125I- labeled
viruses can be adsorbed by standardized
layers of such adherent cells-typically
species of nonpigmented pseudomonads
and Bacillus-and be used to determine
the most avid organisms. Similarly nonad-
herent isolates should also be characterized
for their avidity for metals and viruses.
An effort should be made to extract
relatively large quantities of glycocalyx
from different types of sludge solids and
to determine the feasibility of using it to
adsorb viruses, heavy metals, organome-
tallic compounds and selected materials
from the items on the USEPA hazardous
substances list. It may be possible to bind
the sticky polymers to beads, chips, plates,
and so on, and to use them as a means of
scrubbing treated effluents free of viruses
or other hazards.
Purified glycocalyx should be isolated
form high- and low-avidity isolates and
electrophoretic, biochemical and/or serc-
logical comparisons made. Klebsiella
pneumonias is frequently found in sludge
solids. The chemical structures of Kleb-
siella pneumoniae serotypes are well
known and would be useful models.
The fate of 0.1 to 0.5 ^im polyhydroxy-
butyrate granules in activated sludge treat-
ment needs to be described. It is unclear
whether they are digested during sewage
treatment or if they end up in the effluent
as virus carriers. The kinetics of virus
removal need to be studied in the laboratory
with activated sludge units which have
been heavily seeded with the high avidity
bacteria. Variables such as pH, metal ion,
temperature, aeration rate, C/IM ratio, etc.,
need to be studied systematically.
The importance of ciliates as potential
vectors for viruses should be investigated
further using both pure cultures of Tetra-
hymena and wild mixed types isolated by
flotation methods from sludges. Further-
more, an attempt should be made to follow
the fate of the ciliate-virus combinations
up the food chain. Autoradiographic
methods can be used to see if the double-
labeled virus on ciliates or Zoogloea are
transferred to higher forms such as ne-
matodes, rotifers, and fish. The cytology
and possibly the cytopathology of the host
recipients will be intriguing. The demon-
strated capacity of virus to be transferred
to cultured protozoans as well as wild
ciliates opens up new and exciting possibil-
ities for vector transmission, virus altera-
tion, and kinetic studies of virus removal.
Certainly critical studies must be made of
the virus types which can be ingested and
their survival rate. Numerous questions
remain. What is the longevity of viruses
relative to the protozoa's life cycle and
activity? Is the virus actually ingested or
merely adsorbed to the pellicle? And
finally, the questions which originated the
study. And protozoa actually responsible
for the irreversible loss of viruses during
waste treatment? What percentage of the
1 x 104 PFU/L commonly found in sewage
are ingested by the ciliates?
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James E. Smith is with Syracuse University, Syracuse, NY 13210.
Robert S. Safferman is the EPA Project Officer (see below).
The complete report, entitled "Quantitation of Viruses in Waste and Other
Waters," (Order No. PB 83-190 306; Cost: $ 11.50, subject to change) will be
available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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