• TABLE OF CONTENTS
HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 Abstract
 Introduction
 General materials and methods
 Laboratory studies of virus survival...
 Development of methodology for...
 Monitoring of indigenous enteroviruses...
 Further laboratory studies on the...
 Conclusions
 Composition of media and solut...
 Bibliography
 Biographical sketch
 Copyright














Title: Fate of viruses during aerobic digestion of wastewater sludges
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Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
        Page iv
    Table of Contents
        Page v
        Page vi
    Abstract
        Page vii
        Page viii
        Page ix
    Introduction
        Page 1
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    General materials and methods
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    Laboratory studies of virus survival during aerobic and anaerobic digestion of sewage sludge
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    Development of methodology for recovery of viruses from aerobic sludges
        Page 71
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    Monitoring of indigenous enteroviruses in aerobically digested sludges at two wastewater treatment plants in Gainesville, Florida
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    Further laboratory studies on the survival of viruses in aerobic sludges
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    Conclusions
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    Composition of media and solutions
        Page 202
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    Bibliography
        Page 207
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    Biographical sketch
        Page 215
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    Copyright
        Copyright
Full Text













FATE OF VIRUSES DURING AEROBIC DIGESTION
OF WASTEWATER SLUDGES










By

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


1984











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.















ACKNOWLEDGEMENTS


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.


iii










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


Page

ACKNOWLEDGEMENTS..................................... iii

ABSTRACT ................................ ................ vii

CHAPTER

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

Introduction................................ 31
Literature Survey.......................... 33
Materials and Methods...................... 37
Results and Discussion.................... 47
Conclusions.................................. 68












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

Introduction.............................. 113
Literature Survey .......................... 114
Materials and Methods..................... 119
Results and Discussion..................... 120
Conclusions................................ 150

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

By

Phillip Robert Scheuerman

April, 1984



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

remove viruses.

The survival of virus during aerobic stabilization of

wastewater sludges was studied using laboratory scale


vii










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


viii










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.














CHAPTER I
INTRODUCTION

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





2




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.


A. BIOLOGICAL
1. Anaerobic digestion
2. Aerobic digestion
3. Composting

B. CHEMICAL
1. Lime
2. Chlorination

C. PHYSICAL METHODS
1. Sludge thickening
a. Gravity
b. Flotation
c. Centrifugation

2. Sludge conditioning
a. Chemical
b. Heat
c. Freezing

3. Sludge dewatering
a. Sand beds
b. Vacuum filtration
c. Centrifugation
d. Filter press
e. Belt filters
f. Screens

D. FINAL DISPOSAL
1. Incineration
2. Wet oxidation
3. Land Spreading
4. Landfill of sludge
5. Landfill of incineration ash
6. Pyrolysis


1975 and Clark et al., 1971.


Sources: EPA,











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

day.

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%

(EPA, 1975).

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

biological pathogens.

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.,

1979).

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.


ORGANISM


DISEASE


1. PARASITES
Ascaris lumbricoides
Trichuris trichiura
Taenia sp.
Hymenolepis nana
Giardia sp.
Balantidium coli
Entamoeba histolytica
Toxocara canis

2. BACTERIA
Salmonellae


Shigellae

Escherichia coli

3. VIRUSES
Poliovirus


Coxsackievirus group A




Coxsackievirus group B


Ascariasis'
Trichuriasis
Cysterococosis
Taeniasis
Giardiasis
Balantidiasis
Amoebiasis
Visceral larval


migrans


Typhoid fever
Salmonellosis

Shigellosis

Gastroenteritis


Paralysis
Aseptic meningitis

Herpangia
Aseptic meningitis
Paralysis
Respiratory illness

Pleurodynia
Aseptic menigitis
Pericarditis
Myocarditis
Congenital heart anomalies
Nephritis










Table 1-2. Continued


ORGANISM DISEASE


Echovirus Respiratory infection
Aseptic meningitis
Diarrhea
Peri'carditis
Myocarditis

Reovirus Respiratory infection
Gastroenteritis

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.



Parasites

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

people.



Bacteria

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.



Viruses

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


1955-1956

1965

1966


1966

1967

1967

1969

1971

1971

1972

1973

1976

1978

Adapted f


Delhi, India 28,745

California 2,500

New Jersey 13,000
and Pennsylvania

France 427

New York 7

West Virginia 700

Massachusetts 90

Tennessee 129

Alabama 50

California 26

Wisconsin 26

China <1000

Vermont 2,000


Municipal
system
Public water

Semipublic
water

Public water

Shellfish

Ice

Public water

River water

Semipublic
water
Lake water

Shipboard
water
Well water

Public water


Hepatitis

Gastroenteritis

Gastroenteritis


Hepatitis

Gastroenteritis

Gastroenteritis

Hepatitis

Hepatitis

Hepatitis

Gastroenteritis

Gastroenteritis

Hepatitis

Gastroenteitis


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.














CHAPTER II
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

period.



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

(1938).










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 Assays



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

hours.










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

370C.



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.


Digestion
Sludge Sludge time Additional
Plant designation* type (days) treatment


Campus plant UML Mixed liquor ---
(University of UDA Aerobically 39
Florida) digested

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
dewatered
Pensacola Fl. PDAN Anaerobically 60 Conditioned
Main Street digested with Magnifloc
(dewatered) 2535C and then
dewatered
Montclair and LAG Lagooned ---
Main Street
(Pensacola Fl.)

* 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.















CHAPTER III
LABORATORY STUDIES OF VIRUS SURVIVAL DURING
AEROBIC AND ANAEROBIC DIGESTION OF SEWAGE SLUDGE



Introduction



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.



Primary* Temperature
Dissolved oxygen
Detention time
Sludge source

Secondary Total solids
Volatile solids
Hydrogen ion (10-pH)



* Primary parameters were controlled at set levels for
each run, secondary parameters were monitored for
comparison.










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.



Literature Survey



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

Chapter 6.



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.














Sampling P4


Airstones


Thermometer

- Heater














Figure 3-2. Diagram of the laboratory scale anaerobic
digester used in this study.





























Gas Collection System










--Feed Port





K Stirrer


Sampling Port


Heater


Bath











(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

plant.

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.


Hydrogen
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
Echovirus 1
Rotavirus SA-11


32.4
31.4
34


15.5
ND
ND


9.3
ND
ND


0.4
0.3
0.05


(6.4)
(6.5)
(7.3)


Table 3-2.











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.
ND=not done.


2.6
ND
8.9
ND
6.1
4.2
ND


0.05 (7.3)
2 (5.7)
1.24 (5.9)
7 (5.2)
0.96 (6.0)
3.26 (5.5)
4.6 (5.3)


6
7
8
9


--


-- -- -- -- -- -- -- -- -- -- -- -- -- -- -- ----------------------------


- - - -


__










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











Table 3-3.


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
plant

Gainesville 28.7A 4.4C 8.9A 6.1A 0.71A
Main Street

Gainesville 28.1B 5.6B 12.5A 4.2B 0.87A
Kanapaha


* 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
(5.3)
17.6 5.2A 7.9B 5.7A 1.1 x 10-6B 0.5B
(6)

5.5C 5.8A 19.8A 5.2A 5.5 x 10-7B 0.21C
(6.3)


different at


* 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).

























































DAYS











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.















0.0










ADAYS

J \0C

.- 0-o 5C




-2.0
1 2 3 4 5 6 7 8 9 10 11 12
DAYS










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










Table 3-5.


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
(5.3)

32.4A 0D + 15.5 9.3A 3.8 x 10-7B 0.33A
(6.4)

27.9B 0.88C 8.6C 2.6C 1.4 x 10-7B 1.03B
(6.9)

28.28 2.7B 20.6A D++ 2.3 x 10-6AB 0.91B
(5.6)


* 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.


















E
-i
=
La.


-I
-1.0

z








-2.0


-5.0 mg/L 02

0-0 ANAEROBIC




1 2 3 4 5 6 7 8 9 10

DAY












Figure 3-6. Daily change in poliovirus during aerobic
sludge digestion at 1.0 and 5.0 ppm dissolved
oxygen.
0--0 5.0 mg/l 02
.----6 1.0 mg/l 02





























1.0
LA.I


C.-
E

,.
e







I -2.0
ej


% \


I
'I

I1

'I


-3.0 1 1 1 --1-
1 2 3 4 5 6 7 8 9 10


DAY










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











Table 3-6.


Effect of detention time on Poliovirus (type 1, Lsc) survival in laboratory
scale digesters.*


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
p=0.05.










digestion and the digestion temperature used (280C) was that

temperature where detention time begins to decrease in

significance.



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.


Table 3-7.










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)

Volatile -0.79
solids (0.11)

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.


Table 3-8.










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

enteroviruses studied.











Table 3-9.


Comparison of virus survival during aerobic
digestion of wastewater sludges at various.
conditions.


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
1
Coxsackievirus -0.46A 27.4 4.8
B3

Rotavirus -0.43A 28.0 5.8
SA-11


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
SA-11


Poliovirus -0.33C 32.4 +
(type 1, Lsc)

4 Echovirus 1 -0.53C 31.4

Rotavirus -0.52C 34.0
SA-11


* Treatments described in Table 3-2.
** Means with the same letter for each treatment are not
significantly different at the p=0.05 level.
Anaerobic digestion.









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

sludges.



Conclusions



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.


Treatment*


Percent virus
in the sludge
supernatant
(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

digestion.














CHAPTER IV
DEVELOPMENT OF METHODOLOGY FOR RECOVERY OF
VIRUSES FROM AEROBIC SLUDGES



Introduction



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.



Literature Survey



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


3% beef-extract
blending
sonication

Glycine buffer
(0.05M),pH 11.5 +
magnetic stirring


3% beef-extract
pH 9.0,sonication
magnetic stirring
or freon extraction


Organic
flocculation


Organic
flocculation
filtration of floc
supernatant

Polyethylene glycol
hydroextraction


Indigenous



Poliovirus type 1
Coxsackie B3
Echovirus 7


Indigenous


Undigested and
anaerobically
digested

Mixed liquor and
Aerobically
digested


Anaerobically
digested and
dried sludge


Glass et al. (1978)



Hurst et al. (1978)




Wellings et al. (1976)


3% casein None
hydrolysate
3% Lactalbumin
hydrolysate
3% beef-extract
3% fetal calf serum
wrist action shaking


Indigenous


Raw


SSattar and Westwood (1976)











Acid precipitation None
10% beef-extract
pH 7.0, Alum
precipitation
and Tris pH 9.0
elution with 10%
beef-extract pH 7.0


Indigenous


Raw,
anaerobically
digested, and
sludge-soil
mixture


Nielsen and Lydholm
(1980)


Distilled water
+ blending


None
(direct
innoclulation)


Earle's balanced
salt solution
2% FCS pH 9.5 +
blending

10% beef-extract
in McIlvaine buffer
pH 7.0 + magnetic
stirring


Bentonite adsorp-
tion following
tryptose phosphate
broth elution


None


None


None


Indigenous
Poliovirus type'1


Indigenous


Raw,
anaerobically
digested, and
lagooned

Anaerobically
digested,
primary, and
mixed liquor


Poliovirus type 1


Indigenous


Turk et al. (1980)
Moore et al. (1979)


Lund (1973)


Subrahmanyan
(1977)


Mesophilic
anaerobically
digested and
thermophilic
anaerobically
digested


Berg and Berman
(1980)











Table 4-1 continued


Elution Concentration Sludge
Method Method Virus Type Reference


Phosphate buffered
saline pH 9.8


Beef-extract in
citric acid buffer
pH 7.0 + magnetic
stirring



Fetal calf serum
in
saline (10%)
pH 7.2


None



None


Indigenous



Indigenous


None


Indigenous


Undigested and
anaerobically
digested

Waste activated,
anaerobically
digested, and
thermophilic
aerobically


Lagooned and
anaerobically
digested


Vasl and Kott
(1981)


Kabrick et al.
(1979)





Sattar and Westwood
(1979)












0.1% SDS + 0.05M
glycine pH 7.5
(3 elution steps)


3% beef-extract Poliovirus type 1 Anaerobically
organic flocculation digested
(3 subsequent
flocculation
steps)


Abid et al. (1978)


0.4M urea + 0.1M
lysine pH 9.0 +
magnetic stirring


Alum flocculation
elution with
beef-extract
(5%) and
subsequent organic
flocculation


Poliovirus type 1
Coxsackie B3
Echovirus type 1


anaerobically
digested and
aerobically
digested


Farrah et al.
(1981B)













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

al., 1976).

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
sludges.*



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
types.


* Part of this evaluation process was performed while
testing methods for inclusion as an ASTM standard
method.









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).


Chemicals

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

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

Table 2-1.



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

assay.

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

virus.

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-

19:24:04:04/05.

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

virus.

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

glycol hydroextraction.

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

Urea-lysine procedure.



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











Table 4-4.


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




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