SAND76-0350
Unlimited Release
Waste Resources Utilization Program
Interim Report
June 30, 1976
Waste Management and Environmental Programs Department
:unded
Municipal Environmental Research Laboratory
U. S. Environmental Protection Agency
Cincinnati, Ohio
Division of Nuclear Research and Applications
U. S. Energy Research and Development Administration
Washington, D.C.
Prepared by Sandia Laboratories, Albuquerque. New Mexico, 87115
and Livermore. California 94550 for the United States Energy Research
and Development Administration under Contract AT (29-1) 789
Reprinted January 1977
-------
Issued by Sandia Laboratories, operated for the United States Energy
Research and Development Administration by Sandia Corporation,
NOTICE
This report was prepared as an account of work sponsored by
the United States Government. Neither the United States nor
the United States Energy Research and Development Admini-
stration, nor any of their employees, nor any of their contrac-
tors, subcontractors, or their employees, makes any warranty,
express or implied, or assumes any legal liability or responsibility
for the accuracy, completeness or usefulness of any information,
apparatus, product or process disclosed, or represents that its use
would not infringe privately owned rights.
-------
SAND76-0350
Unlimited Release
Printed July 1976
Reprinted December 1976
Reprinted January 1977
WASTE RESOURCES UTILIZATION PROGRAM
INTERIM REPORT
JUNE 30, 1976
Waste Management and Environmental
Programs Department 5440
Sandia Laboratories
Albuquerque, NM 87115
This work was funded by the Municipal Environmental
Research Laboratory, U. S. Environmental Protection
Agency, Cincinnati, Ohio, and the Division of Nuclear
Research and Applications, U. S. Energy Research and
Development Administration, Washington, D. C.,
Interagency Agreement AT(29-2)-3536/EPA-IAG-D6-0675.
Printed in the United States of America
Available from
National Technical Information Service
U. S. Department of Commerce
5285 Port Royal Road
Springfield, Virginia 22151
Price: Printed Copy $6.00*; Microfiche $3.00
*Add $2.50 per copy for foreign price
3-4
-------
PREFACE
This is an interim report on the effects of the combined
use of heat and ionizing radiation (thermoradiation) as a
treatment for ridding sewage sludge of pathogenic organisms
as well as its effect on the physical-chemical properties.
This activity couples two major environmental problems,
disposition of human and of nuclear waste, in an attempt to
provide a framework in which both will become useful resources.
This combined treatment might be chosen to inactivate both
heat labile (but possibly radiation resistant) and radiation
labile (but possibly heat resistant) organisms. The cost-
effective analyses of such a treatment are being examined.
Sludge treated with thermoradiation offers considerable
potential for use as a fertilizer in agriculture or a soil
conditioner for land reclamation free of the potential health
hazards associated with conventional methods of land disposal.
Treated sludge may also provide a low-cost substitute for
high-nutritional components in ruminant diets.
In order to determine the feasibility of treating sewage
sludge with thermoradiation, a number of parameters have to
be examined. These objectives include determining (1) the
amount of thermoradiation needed to inactivate the major
biological systems (i.e., bacteria, viruses, and parasites)
found in sludge that are potentially harmful to humans; (2)
the cost of such a treatment versus the value of the benefits
from sludge usage; (3) any additional benefits to physical-
chemical properties accruable by a treatment process involving
irradiation; and (4) the design of optimal treatment facilities
whereby actual sludge assessment could be determined.
5-6
-------
CONTENTS
Page
PREFACE 5
IRRADIATION SYSTEMS 13
Milliliter System 14
Liter/Minute Flow-Through System 16
Proposed Pilot Plant 21
Cesium-137 Trailer 28
BACTERIOLOGY 3Q
Introduction 30
Experimental 32
Results 35
Summary 66
VIROLOGY 70
Poliovirus Inactivation in Sludge 70
Heat Inactivation of Poliovirus in Raw and
Anaerobically Digested Sludge 92
PARASITOLOGY 107
Introduction 107
Experimental 109
Results 112
Summary H5
EFFECTS OF HEAT AND IRRADIATION ON PHYSICAL/CHEMICAL
PROPERTIES OF SEWAGE SLUDGE 118
Introduction 118
Experimental 119
Results 122
Primary Digester Sewage Sludge 124
Undigested Sewage Sludge 130
Summary 130
-------
CONTENTS (cont)
Page
COST BENEFIT ANALYSES 131
CONCLUSION 139
FUTURE WORK 140
REFERENCES 144
FIGURES
Page
1 Schematic Drawing of Milliliter System 15
2 Flow-Through Thermoradiation System 18
3 Irradiator 19
4 Schematic of Pilot Plant Irradiation
Room Layout 24
5 Proposed Sludge Processing Flow Schematic
for Pilot Plant 25
6 Heat-up Profiles for Remote Sampling System
(70° and 40° C) 33
7 Profile for "Fast-rise" Chamber Heat-up 34
8 Radiation Inactivation of Coliforms at
20° C and 30 krads/minute 36
9 Inactivation of Coliforms at 50° C and
30 krads/minute 37
10 Heat Inactivation of Coliforms 38
11 Inactivation of Coliforms at 65° C and
30 krads/minute 40
12 Coliform Survival 41
13 Inactivation of Fecal Strep and Coliform
Bacteria by Heat at70°C 42
14 Radiation Inactivation of Fecal Strep at
20° C and 30 krads/minute 43
15 Heat Inactivation of Fecal Strep 44
16 Heat and Radiation Inactivation of Fecal
Strep 46
-------
FIGURES (cont)
Figure Pag_
17 Fecal Strep Inactivation at 60° C and
30 krads/minute 47
18 Fecal Strep Survival 48
19 Inactivation of Coliform Bacteria at
Different Dose Rates 49
20 Inactivation of Fecal Strep Bacteria at
Different Dose Rates 50
21 Inactivation Profiles for Radiation Treatment
of Fecal Strep in Saline Inoculated with
Broth (•) and in Sludge Inoculated with
Broth (Q) . Dose Rate *70 krads/minute. 52
22 Thermoradiation Inactivation Profile for
Fecal Strep in Inoculated Saline (•) and
Inoculated Sludge (Q). Dose Rate is 15
krads/minute. 53
23 Growth Profile for Sterile Sludge Inoculated
with Low Levels of Coliform Bacteria
(Untreated Sludge) 55
24 Growth Profile for Sterile Sludge Inoculated
with Low Levels of Fecal Strep Bacteria
(Untreated Sludge) 56
25 Inactivation Curve for Salmonella Species
Using Both Hektoen Enteric (HE) and
Salmonella-Shigella (SS) Agars at 23° C. 59
26 Radiation Inactivation Curves for Salmonella
Species in Different Sludges at 23° C. 60
27 Thermoradiation Inactivation of Salmonella
Species, With and Without Oxygenation at
50° C. 62
28 Heat Inactivation for Salmonella 63
29 Effect of Oxygenation on Coliform Inacti-
vation Rates at 23° C 64
30 Oxygenation Effect on Thermoradiation
Inactivation of Coliforms at 55° C 65
31 Oxygenation Effect on Salmonella Inacti-
vation Curves (Inoculated Sludge) at
23° C 67
32 Effect of Sludge on the Sedimentation
Coefficient of Poliovirus. 81
-------
FIGURES (cont)
_
33 Isolation of Poliovirus Particles by Density
Gradient Centrifugation Following Incu-
bation in Sludge. 83
34 Density Gradient Centrifugation of Poliovirus
RNA from Particles Recovered from Sludge 85
35 SDS-polyacrylamide Gel Electrophoresis
Pattern of Poliovirus Proteins from
Particles Recovered from Sludge 88
36 Effect of Sludge on the Rate of Heat Inacti-
vation of Poliovirus 95
37 Sedimentation Profiles of Radioactively-
Labeled Poliovirus Before and After Heat
Treatment in Anaerobically Digested Sludge 98
38 Survival of Poliovirus After Heat Treatment
in Various Concentrations of Anaerobically
Digested Sludge 100
39 Heat Inactivation of Ascaris lumbricoides Ova 113
40 Thermoradiation Inactivation of Ascaris
lumbricoides Ova
41 Reduction in Embryonation of Ascaris
lumbricoides Ova in Sewage Sludge (5
Percent Solids) and Saline at 23° C 116
42 Apparatus for Settlability Measurements 120
43 Filtration Apparatus 121
44 Settlability Profiles for Heat, Radiation
and Thermoradiation 123
45 Radiation Effects on Filterability 125
A. Typical Time/Unit Volume versus
Volume Plot
B. Normalized Specific Resistance
versus Dose
46 Thermal Effects on the Specific Resistance
of 1, 3, and 5 Percent Solids — Digested 126
47 Radiation and Thermoradiation Effects on the
Specific Resistance of 5 Percent Solids —
Digested 127
10
-------
TABLES
Table p^
I Sensitivities of Various Microorganisms
to Heat and Radiation 31
II Results of "Growth" Experiments in Sludge
for Various Initial Conditions. Samples
were Incubated at 35° C 57
III D-Values (Treatment Per Log Reduction) for
Coliform Bacteria at Various Temperatures 68
IV D-Values for Fecal Strep Bacteria at Various
Temperatures 69
V D-Values for Salmonella Species Added to
Sewage Sludge 69
VI Recovery of Plaque-forming Units after
Mixing Poliovirus Strain CHAT with
Digested Sludge for 15 Minutes at Room
Temperature 7 2
VII Recovery of Poliovirus in Digested Sludge
Supernatant upon Reextraction of Sludge
Solids 74
VIII Recovery of Poliovirus (Strains Mahoney and
712) after 15 Minutes in Digested Sludge 75
IX Recovery of Poliovirus Plaque-forming Units
from Digested Sludge as a Function of the
Time of Incubation 77
X Recovery of Poliovirus from Digested Sludge
after Incubation for 3 Days at 20° C by
Reextraction of Sludge Solids 79
XI Infectivity of Poliovirus RNA Extracted from
Particles Incubated in Digested Sludge 86
XII Effect of Proteolytic Enzymes and Ribonuclease
on Poliovirus Infectivity 90
XIII Effect of Raw Sludge on Poliovirus Infectivity 91
XIV Inactivation of Poliovirus by Various Fractions
of Digested Sludge During 3 Days at 28° C 93
11
-------
TABLES (cont)
Table
XV
XVI
XVII
XVIII
XIX
XX
XXI
Recovery of Radioactively-Labeled Poliovirus
Strain CHAT in Digested Sludge Supernatant
after Heat Treatment 97
Effect of Sludge Concentration on Heat
Inactivation on Poliovirus 101
Survival of Poliovirus Strain CHAT after Heat
Treatment in Fractionated Sludge 103
Effect of Virucidal Activity on Heat Inacti-
vation of Poliovirus in Raw Sludge 104
Percent Embryonation in Selected Media ill
Inactivation of Ascaris lumbricoides Ova 117
Specific Resistance (1012 m/kg) of 5 Percent
Solids-Digested 129
12
-------
IRRADIATION SYSTEMS
Progress reported in this section relates to the devel-
opment of a series of thermoradiation treatment systems for
use with sewage sludge. The first of these, the "milliliter
system" was designed primarily to facilitate small-scale
biological experimentation. It involves an agitated batch
process of a small quantity of sludge in which process
parameters are highly controlled. In its design there was
no intent that any ultimate treatment plant should operate
in the same way.
Indeed, the philosophy guiding ultimate treatment plant
design has been one favoring a real-time flow-through
processing system in which sludge residence time is a matter
of only a few minutes. In order to progress from a small
experimental batch processer to a full-scale treatment
facility, two intermediate steps were planned.
The first was the development of a real-time flow-
through thermoradiation system capable of processing one to
several liters of sludge per minute (the "liter system").
This system was intended as both a research facility for
studying biological effects and engineering characteristics
and as a system capable of processing sufficient sludge for
studies of the application of sludge to land and to animal
feeding programs.
The second step is to pilot plant level capable of
processing up to 20,000 gallons of sludge per day (some
~15 gallons per minute).
13
-------
Milliliter System
The milliliter thermoradiation system was developed to
study the microbiology of sewage sludge for the purpose of
establishing heat, radiation and thermoradiation treatment
criteria for the elimination of pathogens.
The source elements used for the construction of the
irradiator for this system consist of 26 pins of cesium-137
chloride that are double-encapsulated in stainless steel.
Each pin is 2.79 cm in diameter by 30.5 cm in length. The
total activity of the 26 pins is 208 KCi. The irradiator
that uses these pins is illustrated in Figs. 1 and 3. The
pins are placed two deep in an annular array so as to produce
a fairly uniform field in the interior of the annular region.
The effective volume of uniform field strength is 10 cm in
diameter by 40.6 cm in length. The field strength in air in
this region is approximately 850 rads/sec and is fairly
uniform.
The apparatus used to heat the sample of sludge is
shown schematically in Fig. 1. A 160 m£ sludge sample is
heated in a coaxial assembly with heat being applied to both
the inner and outer annular surface of the liquid. The
inner coaxial surface is a plunger that is alternately
raised and lowered to provide mechanical agitation. Water
from a water bath is recirculated through the outer jacket
and the plunger. A sample temperature versus time curve
for the apparatus is shown in Fig. 6. For most temperatures,
the heat-up time is several minutes.
Six spring loaded syringes that are activated sequen-
tially by remotely located timers take the sludge samples
through stainless steel tubing to an ice cooled reservoir at
selected times.
14
-------
WATER
SLUDGE SAMPLE
SLUDGE
^MB«l
••
n
\
•^
<;
s
s
s
s
s
s
N
S
N
S
S
s
S
S
S
V
s
s
\
s
s
s
s
s
s
s
s
s
s
V
s
s
s
s
k \
^
s
s
V
s
V
s
s
s
s
s
s
s
S
s
N
S
N
^
•
^
s
s
s
s
s
s
s
s
s
s
s
s
>
s
s
s
s
s /
I <*- WATER IN
CESIUM-137 RODS
Figure 1. Schematic Drawing of Milliliter System
15
-------
The apparatus described above is used in the following
manner in an experiment. First, the water bath is turned on
and the temperature of the water jacket allowed to reach a
predetermined level with the water recirculating. Control of
this temperature can be exercised within ±0.2° C over the
range from approximately 5° C to 90" C. The irradiator is
stored in approximately 6 m of water. To initiate the test,
the irradiator is raised into a concrete shielded room. The
heating apparatus described above is surrounded by the annular
shaped irradiator. As the irradiator is raised, the sludge,
or other type sample, is injected into the coaxial heating
assembly. The temperature is elevated to the preset value
before the sample begins receiving treatment (dose rate is
30 krads/min). Lead shields allow this dose rate to be
reduced by a factor of four when desired. At preselected
times, the sludge is remotely sampled using electrical
signals fed in by timers located outside the shielded room.
After the test, the irradiator is lowered into the pool and
the sludge samples removed for analysis.
This apparatus has been the mainstay of the biological
experimentation using thermoradiation at Sandia Laboratories.
Hundreds of experiments on fecal strep, fecal coliform,
Salmonella and poliovirus have been performed with this
system.
Liter/Minute Flow-Through System
The flow-through system was designed to treat larger
quantities (10,000 to 20,000 I) of sewage sludge. After
treatment it was then dried to -25 percent solids. The
dried sewage sludge is being used for animal refeeding
experiments and fertilizer trials at New Mexico State
University. The information gained from this system aided
in the design of a pilot plant for the City of Albuquerque.
16
-------
An effective pasteurization treatment was developed
during experimentation using the milliliter system. Sludge
is irradiated to 150 krads at a temperature of 65° C with a
5 minute residence time in the irradiator. Hardware was
designed using the irradiator described in the last section
to treat approximately 1 A/rnin to these specifications. The
irradiator and the treatment unit are shown in Fig. 3.
A flow schematic for the system is shown in Fig. 2.
Sludge from Albuquerque's Water Reclamation Plants No. 1 and
2 was brought by truck in containers to the irradiation area.
The sludge was then transferred in the plenum shown at the
start of the system (Fig. 2). A recirculating pump was
used to stir the sludge and to provide a small positive
pressure for the inlet to the tubing pump that metered the
sludge flow. The sludge was then pumped through counterflow
heat exchangers. A 36 kW hot water heater was the heat
source for the heat exchangers. Heated sludge was then
pumped into the irradiator chamber, through the irradiator,
and back out of the chamber.
The counterflow heat exchangers were fabricated from
1.25 cm stainless steel tubing and 1.9 cm galvanized pipe.
At the low flow rate of the sludge (~1 fc/min) a point of
concern was the adequacy of heat transfer across the
stainless steel tubing. The equation describing an idealized
counterflow heat exchange is given by
G = UA
FAt2 - Ati 1
Lln(At2/At1)J
where:
17
-------
PRESSURE
SWITCH
SLUDGE
PUMP
VALVE
e-
PUMP
PRESSURE
HEAT
EXCHANGER
HEAT
EXCHANGER
HEAT EXCHANGER
WATER HEATER
PUMP
WASTE
BARREL
UNTREATED SLUDGE
PUMP
SLUDGE
PRESSURE
SLURRY
VALVE
TREATED
SLUDGE
-0—-
N,^ VALVE
IRRADIATOR
PUMP
PREHEATING
WATER HEATER
Figure 2. Flow-Through Thermoradiation System
-------
2
90
3
HES
i
i
&
\-
j
1
I i
,
i-
k^_ "5
«*al Jb
11 1
1
.
I •
Vf
11
" ^_J "• **
g^^'^
I -^
^
•~-->
\—r^
F^
•Jl
Sy
\
A
1
1
1
^13 CESIUM TUBES
(26 CESIUM CAPSULES)
Figure 3. Irradiator
19
-------
G = heat transfer rate for the entire heat exchanger
U = heat transfer rate/unit area - unit temperature
A = area of exchanger
A^ = temperature difference at one end of exchangers
At2 = temperature difference at other end of exchangers
For a flow situation similar to what was used in practice,
G, Atlf and At2 were measured and A was taken to be the area
of the stainless steel tubing. The result
U = l.l cal/°C - cm2 - min
was obtained. For very turbulent flow, one can obtain a
theoretical maximum of
U = 28.6 cal/°C - cm2 - min
for stainless steel. Therefore the low flow rates with con-
sequent nonturbulent flow, quite effectively reduce the heat
transfer in heat exchangers, and this effect must be accounted
for in plant design. The easiest way to correct the situation
is to increase the area of the heat exchangers, although later
the problem of increasing U will be addressed. Using ex-
changers of this type, heat recoveries of over 50 percent
have been measured at flow rates similar to those of the
flow-through system.
A fiberglass holding tank held the sludge until it was
carried to the centrifuge. The sludge was then dewatered
using a Sharpies P-660 Super-D-Canter Centrifuge. Polymers
from Hercofloc were used to enhance dewatering by the cen-
trifuge. The moist cake was then dumped into drying tanks
for final drying.
20
-------
Approximately 24,000 liters of sewage sludge (both
digested and raw) have been treated with the thermoradiation
system described above.
In addition to the biological inactivation parameters
studied with this system, it has provided sufficient quan-
tities of treated sludge for some physical and chemical
studies.
Proposed Pilot Plant
The progression from "bench-model" thermoradiation
apparatus to a system of treating larger quantities of sewage
sludge has made the design of a full-scale facility necessary.
The logical choice appears to be a pilot plant located near
an existing sewage treatment plant.
A pilot plant would provide the capabilities for
studying the following: definition of parameters for
demonstration facility; economics of thermoradiation treat-
ment; additional microbiological inactivation and regrowth
studies; sludge dewatering and transport; irradiator design;
cattle feeding and/or land application on a larger scale;
and treatment of digested, undigested and dry sludges.
With the above considerations in mind, a preliminary
pilot plant design has been completed with the help of the
City of Albuquerque, Molzen-Corbin & Associates, and Kramer-
Callahan & Associates. Tentatively, plans are to locate
the pilot plant in Albuquerque at the Waste Water Treatment
Plant No. 2. A 1.4 megacurie cobalt-60 radiation source
valued at $700,000 would be supplied by Sandia Laboratories.
21
-------
A pasteurization treatment using thermoradiation up to
65° C and 500 krads with a residence time in the irradiator
up to 5 minutes will be utilized for wet sludges. Three
different sludge process streams will be possible: (1)
Primary clarifier sludge, (2) Digested sludge, and (3) Waste
activated sludge. All of these would be thickened as neces-
sary to approximately 10 percent solids for the thermoradiation
treatment. The process fundamentals are described below. A
fourth process for treating dried digested sludge is described
separately.
1. Primary Clarifier Sludge
Approximately 40,000 gallons of 4 percent solids primary
clarifier sludge would be treated per day. The sludge will
be initially thickened using a gravity sludge thickener to
8 to 10 percent solids. The supernatant from the thickening
operation will be fed to the waste activated sludge line
which goes to the primary clarifier influent distributor.
The 20,000 gallons/day of thickened sludge will be pumped
through a hot water heat exchanger (from digester heat
supply) to raise the temperature to 50° C. A steam injection
system will raise the temperature another 15° to 65° C, at
which point the sludge will pass through the irradiator.
The treated sludge will then be dewatered to 25 percent
solids using a centrifuge or filter press, or piped directly
to the drying beds.
2. Digested Sludge
Digested sludge can be handled at the rate of 20,000
gallons/day. The process is identical to the primary sludge
treatment except thickening will not be necessary.
22
-------
3. Waste Activated Sludge
The waste activated sludge process is made more difficult
by the low solids content of the sludge (< 1 to 2 percent)
coupled with the ineffectiveness of the gravity thickener.
The activated sludge will first be pumped to the centrifuge
where it will be dewatered to about 5 percent solids. The
supernatant will be pumped to the waste activated sludge
line for return to the plant flow. The thickened sludge will
be pumped to the heat exchangers and then steam injection
used to raise the final temperature to 65° C before being
passed through the irradiator. At this point, the activated
sludge can be dewatered using a filter press or pumped to
the drying beds.
4. Dried Digested Sludge
The dried digested sludge from the drying beds will be
ground to a nominal 1/4" size, then transported to the
processing building by front end loader. The milled sludge
will be put into a hopper on the side of the building from
which a screw type feed conveyor will move the sludge to the
irradiator. After treatment to 1 Mrad, the dried sludge will
be conveyed to a storage bin outside the building.
A preliminary layout of the proposed irradiation facility
is given in Figs. 4 and 5. The design basis for each com-
ponent cost is summarized as follows:
Item Design Basis Installed Cost
Building, Crane Mechanical, electrical, $ 182,000
& Pools 20 ton crane, and ra-
diation tank w/16 ga.
stainless steel lining;
55' x 36'
23
-------
dried sludge storage-
dehumidifier
00
nn
3'x 7'x I-3/4"
hollow metal
doors (both sides)
dried sludge pickup point
55'
d.i. water
heat exchanger
UUI
control panel
8" block walls
filter press
irradiation tank
38'
heat exchanger
centrifuge
data acquisition system
boiler
pump
IRRADIATION ROOM LAYOUT
pump
pump
10' x 10' overhead
^doors (both sides)
Figure 4. Schematic of Pilot Plant Irradiation Room Layout
-------
sludge thickener
primary clarifier
sludge
supernatant
digested
sludge
activated sludge
-0-*
boiler
controls
\hc
steam injector
iheat exchanger
activated
sludge
centrifuge
supernatant
dried
sludge
irradiation
tank
-f- cool ing
-•-filter press
Figure 5. Proposed Sludge Processing Flow Schematic for Pilot Plant
-------
Item
Irradiator
Auger Dried Sludge
Conveyors and
Hopper
Deionized Water
Heat Exchanger
Deionized Water
Makeup System
Centrifuge
Data Acquisition
System for Moni-
toring and Control
Sludge Heat Ex-
changer w/Controls
Sludge Thickener
Boiler and Steam
Injection Equip-
ment w/Temperature
Controls
Filter Press
Oxygen Injection
Equipment with
Controls
Miscellaneous
Piping, Valves,
Meters, Controls,
Electrical
Lighting
Design Basis
By Sandia Laboratories,
in pool
20 yd per day, 50 ft
conveyor with motors,
20 yd 3 storage
Existing unit at Sandia
Laboratories, 22 kw
Existing unit at Sandia
Laboratories, 22 kw
12" bowl with polymer
feed system and motor
control accessories
(Dorr Oliver)
Existing unit at Sandia
Laboratories
20,000 gallons/day to
120° P
Covered, vented to flare,
40,000 gallons/day
ASME 125 psi
15 HP w/pump and controls
1000 Ib dried cake per
24 hrs, polymer-on-
paper precoatings
20,000 gpd to be oxygenated
Installed Cost
$ 100,000
6,000
2,500
500
80,000
30,000
10,000
55,000
6,500
20,000
4,000
30,000
26
-------
I
Item
Pumps and in Line
Sludge Grinder
Primary Sludge
Supply
Digested Sludge
Supply
Waste Activated
Sludge Supply
Waste Sludge
Return
Waste Sludge Line
to Drying Beds
Washwater Supply
Line
Hot Water Supply
and Return Line
Dried Sludge Supply
Paving of Access
Area
Sub-Total:
Design Basis
4 variable speed pro-
gressing cavity pumps
(Moyno) with controls,
1 in-line sludge
grinder
Variable speed pro-
gressing cavity pump,
valves, controls, 4"
pipeline taps
Valves, controls, 4"
pipeline tap, (pump
previously listed)
Valves, controls, 4"
line tap (pump pre-
viously listed)
Valves, 4" line, line
tap (pump previously
listed)
Valves, 4" pipe (pump
previously listed)
Valves, 4" line, line
tap
Insulated 6" line,
valves
Dried sludge grinder
(Royer Model 16)
pavement
Installed Cost
$ 20,000
9,200
5,700
1,900
1,900
13,500
1,200
4,100
4,800
4,000
$ 598,800
27
-------
Subtotal:
$ 598,800
Contingencies @ 10%
59,900
Subtotal:
$ 658,700
Engineering, Legal, Administrative, etc. @ 15% 98,800
Subtotal:
$ 757,500
Inflation (12 months @ 1%) Qn ......
"0,900
TOTAL:
$ 848,400
Cesium-137 Trailer
A trailer mounted cesium-137 irradiator was obtained
during the past year from Sandia Laboratories at Livermore,
California. The source was acquired in order that low dose
rate (-50 rads/sec) thermoradiation studies might be per-
formed .
The gamma source consists of 140 KCi contained in 38
strips; each having an active length of 30.5 cm and an
active width of 2.5 cm. The strips are arranged to form a
rectangular source plaque 56 cm x 69 cm. Each source strip
is doubly encapsulated in stainless steel.
The irradiation unit is a lead-filled steel shell which
contains the fixed source plaque. The product to be irra-
diated is moved into the irradiator by means of a motor
driven shuttle. The shielding from the source plaque is
provided by a series of two mechanically and electrically
interlocked doors.
28
-------
A safety analysis report was prepared for and approved
by the Operational Safety Division of ERDA/ALO before
transfer of the trailer to Sandia Laboratories at Albuquerque,
New Mexico. The irradiator will be tested and made available
for general use within Department 5440.
29
-------
BACTERIOLOGY
Introduction
An excellent survey of the problems associated with the
existence and persistence of pathogens in sewage and sewage
sludge has been published.1
Sensitivities of various microorganisms to heat and to
radiation as reported in the literature are listed in Table I.
The viruses listed tend to be generally radiation resistant
but heat sensitive. The coliforms and staphylococci tend to
be relatively sensitive to either treatment. Streptococci
(and possibly Salmonella) appear to be the most heat and
radiation resistant groups (aside from spores). The majority
of the bacteriological work in sewage sludge has been geared
to either the coliform group, since it is almost a universal
indicator in monitoring effectiveness of wastewater treatment
processes, or to the fecal streptococcus bacteria, which
appear to be the most resistant in terms of both heat and
radiation.
Research areas described in this report include (1)
inactivation rates of coliform and fecal streptococcus bacteria
for treatment by heat and by ionizing radiation, over the
temperature range of 20° C - 70° C; (2) dose rate effects in
inactivation of these organisms in sludge; (3) possible
"protective" effects exerted by the sludge; (4) regrowth
curves in sludge, measured for both coliforms and strep;
(5) inactivation of Salmonella in sludge; (6) effects of
30
-------
TABLE I
Sensitivities of Various Microorganisms to Heat and Radiation
Organism
Adenovirus
Poliovirus
Coliforms
S t aphy lococc i
Salmonella
Streptococci
D-Value*
krads
450
300
20
22
45
120-200
min @ 60° C
0.15
1.5
2
3.3
7.5
15
Reference
2,3
4,5
6
7,8
8,9
10
K
Treatment required per log (base 10) reduction in
population.
U)
-------
oxygenation on the radiation-induced inactivation of coliforms,
Salmonella, and fecal strep.
Experimental
The majority of the inactivation rate studies were made
using the remote sampling system described in section I of
this report. Briefly, spring-loaded syringes allowed sampling
of sludge during a treatment process, or "run." The rise time
(to within a degree of final temperature) following injection
of the sludge ranged from 1 to 2 minutes, depending on tem-
perature (Fig. 6). A "fast-rise" chamber (rise time «20
seconds) was used to measure heat inactivation rates of
bacteria at 70° C (Fig. 7). Regrowth of coliform and fecal
strep bacteria was carried out in spinner flasks in an
incubator at 37° C. Samples were oxygenated simply by
bubbling the pure gas through the sample prior to and during
irradiation. Oxygen concentration was not measured.
Inactivation is measured by the "colony forming ability"
of the bacteria following treatment. Initial counts are
approximately 105 and 104/ml for coliform and fecal strep,
respectively. Appropriate dilutions are plated out on
selective media (agar) in petri dishes. Coliform colonies
are distinguishable as those having a green metallic sheen.11
Fecal streptococci (this plate count technique is a standard
method ) are distinguishable as bright red lens-shaped
colonies embedded in the agar medium. Approximately 150 to
200 plates are required for a single determination of each
inactivation curve. EPA-approved MPN methods13'14 as well
as plate-count techniques (described in the appropriate
section) were used in the determination of Salmonella presence
or inactivation rates in sludge.
32
-------
o
o
. 50 -
<:
LU
Q.
TIME, SECONDS
OJ
Figure 6. Heat-up Profiles for Remote Sampling System (70° and 40° C)
-------
CO
70 k
65
o
o
<
LU
Q_
60 J
" 55 -
50
sample injection
20 30
TIME, SECONDS
40
50
Figure 7. Profile for "Fast-rise" Chamber Heat-up
-------
Results
Inactivation Rates of Coliform Bacteria
Figure 8 shows the radiation inactivation at 20° C of
coliforms by Cs gamma rays. Different symbols indicate
different batches of sludge and/or different "runs." Since
controls were not perfectly consistent on two of the four
runs, normalization to the first inactivation point would
have been justifiable and the data fit better; however, the
slope is the important parameter in this curve. It is seen
that the "D-value" (the absorbed dose required to decrease
the bacterial count by one log, or 90 percent) is approxi-
mately 20 krads/log for coliform bacteria in sludge.
Data from a complete "run", i.e., heat, radiation, and
thermoradiation are shown for one temperature (50° C) in
Fig. 9. These are typical curves. It can be seen that at
this temperature, heat alone contributes very little inacti-
vation over this time scale. The inactivation by combination
treatment exhibits some synergism (approximately one log),
but the effect is considerably less than that observed in
some earlier studies, such as those of E. coli in broth.15
The observed synergism in sludge is consistent, however, and
is seen from 40° to 65° C.
Figure 10 shows heat inactivation curves for coliforms
at temperatures from 40 to 65° C. Only at 55° C and above
does the heat play an appreciable role in inactivation on
this time scale. The results at 60° C and at 65° C are
inconsistent, possibly due to the heat sensitivity of these
bacteria and the heatup profile for the system. However,
radiation results do not indicate this kind of batch-to-batch
variation.
35
-------
D = 20 KRADS / LOG
2 3
TIME, MINUTES
Figure 8. Radiation Inactivation of Coliforms
at 20° C and 30 krads/minute
36
-------
I 1 I I
o
u_
O
>
ID
CO
COMBINATION
11111111111
02468
TIME, MINUTES
Figure 9. Inactivation of Coliforms at
50° C and 30 krads/minute
37
-------
o
C£
u_
o
>
C£.
ZD
CO
TIME, MINUTES
Figure 10. Heat Inactivation of Coliforms
38
-------
Figure 11 shows a plot similar to the 50* C run but at
65° C. The radiation D-value is seen to be approximately
4.5 krads/log at this temperature (versus 20 krads at 20° C).
In Fig. 12, the data for all temperatures are plotted as
surviving fraction at 1 minute (arbitrary time chosen for
demonstration purposes; synergism is in fact somewhat greater
at longer times) versus temperature. While thermoradiation
enhancement of inactivation is greater at higher temperatures,
at lower temperatures there may be some degree of synergistic
behavior.
Figure 13 shows the inactivation of coliforms (and of
strep bacteria) by 70° C heat. This presents a "minimum"
inactivation rate, since there is still a significant heat-up
time for the experimental system. This inactivation rate is
approximately twice that measured using the remote-sampling
system, due to the much slower heat-up profile of the latter.
These data (and others) on coliform bacteria are
summarized at the end of this section.
Inactivation Rates of Fecal Streptococcus Bacteria
Radiation inactivation of fecal streptococci in sludge
is plotted in Fig. 14. It is observed that the radiation
resistance of these microorganisms is approximately six-fold
greater (D-value of about 120 krads/log) than that of the
coliform group. The heat inactivation curves (Fig. 15) show
that the effect of heat alone is minimal below approximately
55° C as with coliforms. The time scale of this plot is
about a factor of three longer than that of the coliform
group (Fig. 10). The inactivation curve for fecal strep by
70° C heat is shown in Fig. 13.
39
-------
o
1—
o
Li-
CS
CO
10
-I
10
-2
10
10
-4
10
10
10
-7
11(111
RADIATION
Bill I I
i I i
30
60
90
TIME, SECONDS
Figure 11. Inactivation of Coliforms at
65° C and 30 krads/rainute
40
-------
5 10' r
o
O
01
IT) rv I i I I
RADIATION
ADDITIVE-*
THERMORAD AT ON
1 I I I I I I 1 1 I
10 u r
65
TEMPERATURE, °C
Figure 12. Coliform Survival
41
-------
10
-5
-6
10
lO'7
0
• CONFORM
O STREP
J I I L
10 15 20 25 30
TIME AT 70°C, SECONDS
Figure 13. Inactivation of Fecal Strep and Coliform
Bacteria by Heat at 70° C
42
-------
0
CO
D«I20 KRADS/LOG
024
6 8 10 12
TIME, MINUTES
18
Figure 14.
Radiation Inactivation of Fecal Strep
at 20° C and 30 krads/minute
43
-------
o
u_
o
>
a:
CO
ICf4
• 70°Cl
• ' 1
i I
I 1
I I
I
I I
I i
I i
I
-
-
i i
2 3
5
TIME, MINUTES
6
7
Figure 15. Heat Inactivation of Fecal Strep
44
-------
These and other data for fecal streptococcus bacteria
are summarized at the end of this section. Again, the
synergism observed with combined treatment (Figs. 16 and 17
show results for 45° C and for 60° C, respectively) is small
but consistent. This is demonstrated in Fig. 18 where the
sum of the heat curves and the radiation curve is shown as
a dotted line. The vertical difference between the dotted
curve and the combined treatment curve is a measure of the
"better than additive" effect. It should be reiterated
that the "four minute" choice is arbitrary and the synergism
is somewhat greater at longer times (for example, refer to
Fig. 17 at 9 minutes).
Dose Rate Effects on Inactivation Rates
Since part of the studies included in this program
involves use of an "outer-ring" irradiation chamber, it was
important to determine whether any dose rate effects would
appear in the inactivation curves. The dose rate was varied
over two orders of magnitude (using cobalt-60, from 1.2 x 10~2
krads/sec up to 1.3 krads/sec) with no apparent change in the
inactivation curves (Figs. 19 and 20). This range certainly
includes any feasible dose rate considered for application
purposes. It is also clear from the slopes of the curves in
Figs. 19 and 20 that, as expected, there is essentially no
difference in the effects of cobalt-60 as compared to those
of cesium-137 (solid lines on these figures), within experi-
mental error.
Sludge "Protective" Effects on Inactivation Rates
Sterilized sludge was inoculated to approximately 106
fecal streptococcus bacteria per milliliter in an attempt to
increase the sensitivity of measurement of inactivation rates
(normal counts in primary digester sludge are ~103 to 104).
45
-------
10
-I
o
C£
u_
o
z:
>
en
10
-2
10
-3
10
-4
I T I I I
i I I I I
HEAT AT 45°c
RADIATION AT 20°C
COMBINED
0
I I I I I I I I I • I I I
8
TIME, MINUTES
10 12 14
Figure 16. Heat and Radiation Inactivation of Fecal Strep
46
-------
o
o
u_
o
>
on
RADIATION (20° C)
20
TIME (MINUTES)
Figure 17. Fecal Strep Inactivation at
60° C and 30 krads/minute
47
-------
i i I I r
CO
10
-i
3
10
-2
o
u_
O
z
>
H)
CO
10
-3
THERMORADIATION
Uv i I I I i I I i ! I I I
20
40
45
50
55
60
65
70
TEMPERATURE
Figure 18. Fecal Strep Survival
48
-------
-I
10
2.0-2
o
i IO'3
10
10
-5
10
-6
COBALT, 1.2 x IQ-2 krads/second
O
COBALT, 1.3 krads/second'
_L
±
J.
0 20 40 60 80
DOSE, KILORADS
O
j.
100 120
Figure 19. Inactivation of Coliform Bacteria
at Different Dose Rates
49
-------
F:—i 1 1 r
10"
o
-------
I
The purpose was two-fold: (1) To compare inactivation rates
in sludge to those in saline, for heat, radiation, and
combined treatment, in order to identify any "protective"
effects of sludge. (2) To use the increased sensitivity of
measurement to determine whether thermoradiation inactivation
curves are linear or if they begin to "tail" or flatten with
longer treatment times.
Figure 21 shows the results of such an experiment for
radiation alone. Apparently there is some protection afforded
by the sludge. Other runs confirm this finding. The sensi-
tivity of the experiment is such that the absence of colonies
on a large number of plates allows ua to state that no "tailing"
occurs with radiation inactivation over at least 6 logs of
strep.
Only one thermoradiation experiment has been performed
at 60° C. Figure 22 shows that there is apparently no sig-
nificant protective effect. There may be some indication of
"tailing" in the sludge samples (the counts at 20 minutes
were significant, while no colonies were observed in the
saline experiment at 20 minutes of treatment); these limited
data, however, are inconclusive.
Bacterial Regrowth Following Thermoradiation Treatment
Following treatment (150 krads at 65° C) of 200 liter
quantities of sludge, coliforms and fecal strep bacteria
were monitored for regrowth. It is unknown whether con-
tamination of the treated material or a residual bacteria
level is responsible for "seeding" the treated sludge. Most
likely both contribute. Contamination and/or regrowth may
present difficulties in any large scale treatment process;
we are presently exploring alternatives to deal with these
problems.
51
-------
10
10
o
o
10
=• 10
CO
0
8 10 12
TIME, MINUTES
Figure 21. Inactivation Profiles for Radiation Treatment of
Fecal Strep in Saline Inoculated with Broth (•)
and in Sludge Inoculated with Broth (Q• Dose
Rate » 70 krads/minute.
52
-------
o
o
Od
>
>
IO'1 L
10
-2
10
-3
10
-4
10
-5
10
-6
10
-7
TIME, MINUTES
Figure 22. Thermoradiation Inactivation Profile for Fecal
Strep in Inoculated Saline (•) and Inoculated
Sludge (O) • Dose Rate is 15 krads/minute.
53
-------
Several experiments were performed in an attempt to
clarify the regrowth phenomenon in treated sludge. Autoclaved
sludge was "seeded" with normal, fresh sludge from a primary
digester, such that initial counts were low. The samples
were stirred and incubated at 35° C and samples were taken
periodically. The results are shown in Figs. 23 and 24 for
coliforms and strep, respectively. The open circles repre-
sent well-aerated samples (open spinner flasks) and the
closed circles represent partially aerated samples (closed
flasks with cotton plugs in the two side parts). It is
clear that, within a day or two, the levels of both strep
and coliform are more than 10 times higher than normal
primary sludge levels.
In addition to these experiments, fecal streptococci
o
were grown in KP streptococcal broth to levels of 10 /ml in
order to determine growth curves under various conditions
of sludge. Among the samples were (1) sterilized sludge
inoculated with broth (initial strep count ~10 /ml), (2)
primary digester sludge inoculated with active broth (initial
strep count ~10 /ml), and (3) primary digester sludge
4
(initial count ~10 /ml). These samples were stirred at
35° C in open spinner flasks. Rapid growth of fecal strep
occurred only in the flasks containing sterilized sludge,
indicating that bacterial competition in untreated sludge
is such that fecal strep growth is not efficient. Table II
shows fecal strep counts versus time under these incubation
conditions for the various samples.
Similar growth experiments on Salmonella species from
sludge have been unsuccessful and are being repeated.
54
-------
CtL
LU
o_
CO
o
o
10
20 30
TIME, HOURS
40
50
Ul
Ul
Figure 23. Growth Profile for Sterile Sludge Inoculated with
Low Levels of Coliform Bacteria (Untreated Sludge)
-------
Ol
10° -
105
10'
to
8
o
10
20 30
TIME , HOURS
40
50
Figure 24. Growth Profile for Sterile Sludge Inoculated with Low Levels
of Fecal Strep Bacteria (Untreated Sludge)
-------
TABLE II
Results of "Growth" Experiments in Sludge for Various
Initial Conditions. Samples Were Incubated at 35° C.
Sample
(a) sterile sludge inoculated
with broth
(b) untreated sludge inoculated
with broth
(c) untreated digester
sludge
Normalized Counts/ml
0 hrs.
1.0
1.0
1.0
24 hrs.
560.0
0.049
0.37
48 hrs.
990.0
0.0069
0.12
72 hrs.
1030.0
0.091
-------
Inactivation Rates for Salmonella from Sewage Sludge
Due to the low counts of these pathogenic bacteria in
sewage sludge (several tens of bacteria per milliliter), it
is virtually impossible to determine inactivation "rates" in
the usual way. A somewhat artificial, yet meaningful, system
was devised. Salmonella species from sludge were grown in
Trypticase Soy Broth to a level of ~109 per milliliter. Small
quantities of this broth were added to sterilized sludge, such
that the starting count of Salmonella bacteria was routinely
10 - 10 per milliliter. Standard inactivation rates were
then determined using these samples. Survival of the bacteria
after inactivation was measured by colony growth on Hektoen
Enteric Agar (HE) which is moderately selective for Salmonella,
or on Salmonella-Shigella Agar (SS), which is a highly selec-
tive medium. Random colonies were routinely checked
biochemically, to insure that there was no interference from
other bacteria; in all cases, these colonies tested positive
for Salmonella. Figure 25 shows that radiation inactivation
rates were not dependent on the type of agar used. Therefore,
due to ease in counting and in colony differentiation, HE
agar was used in most subsequent experimentation. It should
be pointed out that inactivation rates were found to be
essentially the same whether a broth sample was added to
(a) normal, non-sterilized sludge or to (b) sludge which had
been autoclaved for an hour, or to (c) sludge which had been
irradiated to approximately 1.4 Megarads (Fig. 26).
There is some question, of course, as to whether data
obtained using these "artificial" systems can be used to
accurately predict the behavior of the normal Salmonella flora
of digested sludge. As indicated, the species were originally
taken from normal sludge. However, when fecal strep bacteria
were irradiated in a totally analogous system, the inactiva-
tion rate was found to be approximately the same as that
58
-------
0
O - SS AGAR
0 = HE AGAR
50 100
DOSE(KRADS)
150
Figure 25. Inactivation Curve for Salmonella Species
Using Both Hektoen Enteric(HE) and
Salmonella-Shigella (SS) Agars at 23° C.
59
-------
o -2
GO
0-4
IRRADIATED SLUDGE
(INOCULATED)
AUTOCLAVED SLUDGE
(INOCULATED)
XD
NON-STERILIZED'
F SLUDGE (INOCULATED)
i
0
Figure 26.
50 100 150
DOSE(KRADS)
200
Radiation Inactivation Curves for Salmonella
Species in Different Sludges at 23° c.
60
-------
previously published for fecal strep in sewage sludge.16
There is an indication that the broth-grown strep may be
slightly more heat sensitive, but additional studies will be
made to confirm this observation.
Inactivation curves for Salmonella species in sewage
sludge were determined at 50° C as described above. The rate
of inactivation was found to be approximately 50 percent
greater than that at 23° C (compare Fig. 27 with Figs. 25
and 26). Experiments performed several months later gave
inactivation rates for Salmonella (same culture) which were
considerably higher (organisms appeared to be more sensitive)
than the data presented in Fig. 27. Heat inactivation curves
have recently been measured for Salmonella in sewage sludge;
these data are shown in Fig. 28. It is seen that Salmonella
are much more heat sensitive than either coliforms or fecal
strep. This may be due in part to the artificiality of the
system. Again, further studies are under way with strep and
coliforms to determine if they behave similarly under anal-
ogous conditions.
Oxygen Effects on Inactivation Rates
Figures 29 and 30 show inactivation curves for coliform
bacteria in sewage sludge at 23° C and at 55° C, respectively,
for normal runs and for oxygen-saturated samples. For the
latter, oxygen was bubbled through the sludge for about 10
minutes prior to irradiation, and continuously during the
irradiation. It is seen that inactivation at room temper-
ature by irradiation with oxygenation is at least equivalent
to normal (no oxygen) inactivation at the higher temperature.
This observation may be important in achieving inactivation
at lower treatment temperatures. The effectiveness of
oxygenation affords a further enhancement of the rate of
inactivation during thermoradiation at 55° C.
61
-------
>
>
CO
o
O
-7
NORMAL
50 100
DOSE(KRADS)
150
Figure 27. Thermoradiation Inactivation of Salmonella
Species, With and Without Oxygenation at—
50° C.
62
-------
o
o
u.
o
>
GO
o
O
-7
1234
TIME (MINUTES)
Figure 28. Heat Inactivation for Salmonella
63
-------
NORMAL
50 100
DOSE(KRADS)
150
Figure 29.
Effect of Oxygenation on Coliform
Inactivation Rates at 23° C
64
-------
NORMAL
10
20 30
DOSE(KRADS)
40 50
Figure 30. Oxygenation Effect on Thermoradiation
Inactivation of Coliforms at 55° C
-------
Oxygenation appears to be somewhat less effective for
fecal strep bacteria; but, again, the inactivation at room
temperature is as effective with oxygen as is normal thermo-
radiation at 60° C.
Figures 27 and 31 show comparison curves for Salmonella.
As was observed for coliforms and fecal strep, at the selected
temperature, oxygenation is more effective than normal thermo-
radiation treatment for Salmonella.
Part of the enhancement in inactivation rates at higher
temperatures with oxygen must be due to the increased heat
transfer brought about by the bubbling action. This is
demonstrated by the observation that bubbling nitrogen
enhances the inactivation rate somewhat, even though nitrogen
is inert, from a radiation biology standpoint. In room
temperature work, nitrogen tends to displace whatever oxygen
is normally present in the air-saturated samples, and the
inactivation rates generally are slightly less than in normal
runs. Oxygen enhancement of radiation damage in biological
systems is a well-known phenomenon.
Summary
Inactivation rate data for coliforms, fecal strep, and
Salmonella are summarized as D-values for ionizing radiation
at a variety of temperatures in Tables III, IV, and V,
respectively.
These data indicate that radiation, particularly thermo-
radiation, especially in the presence of oxygen, is an
efficient means of disinfection of sewage sludge, from a
bacteriological standpoint.
66
-------
0
NORMAL
vO
50 100
DOSE(KRADS)
150
Figure 31.
Oxygenation Effect on Salmonella
Inactivation Curves (Inoculated
Sludge) at 23° C
67
-------
TABLE III
D-Values (Treatment Per Log Reduction) for
Coliform Bacteria at Various Temperatures
Temperature
(° C)
23
40
45
50
55
60
65
D-Value, krads/log
Normal
25 - 30
25
20
23
10 - 15
15
5
With 02
8
4
68
-------
TABLE IV
D-Values for Fecal Strep Bacteria
at Various Temperatures
Temperature
(° C)
23
40
45
50
55
60
65
D-Value , krads/log
Normal
130 - 135
129
129
109
86
70 - 97
46
With 02
87
32
TABLE V
D-Values for Salmonella Species
Added to Sewage Sludge
Temperature
(° C)
23
50
D-Value , krads/log
Normal
26
19
With 02
13
11
69
-------
VIROLOGY
Poliovirus Inactivation in Sludge
This section is divided into two parts. The first part
deals with the effects of anaerobically digested sludge on
seeded poliovirus at ambient temperatures and below. The
second portion is concerned with the effects of both raw and
anaerobically digested sludge on the rate of poliovirus in-
activation by heat.
Inactivation of Poliovirus in Anaerobically Digested Sludge
Previous studies suggest that some viral inactivation
occurs during anaerobic digestion.17'18 This conclusion is
based primarily on results derived during treatment plant
operation. It is difficult to evaluate these data because
conditions could not be controlled during treatment, the
same material could not be sampled before and after treatment,
and there was no way of determining the efficiency of virus
recovery from either raw or digested sludge. Most of these
technical problems can be avoided by studying the effects of
milliliter quantities of sludge on highly purified,
radioactively-labeled virus. With this procedure, the
original number of infectious virus is clearly established
and the relationship between inactivation and recovery can
be monitored at all times under controlled conditions. Appli-
cation of this method is made here to a study of poliovirus
inactivation in digested sludge.
70
-------
Recovery of Poliovirus from Seeded Sludge—The effects of
digested sludge on poliovirus can be properly evaluated only
if it is possible to account for the entire population of
infectious virus. Because sludge solids have a tendency to
bind virus, thereby creating a potential difficulty in virus
recovery, a method designed to release bound particles after
treatment with sludge was used throughout this entire study.
This method was to sonicate in the presence of the detergent
sodium dodecyl sulfate (SDS). Samples were then either
directly analyzed for plaque-forming units or first centri-
fuged (18,000 g, 20 minutes) to remove large particulate
matter before assaying for infectious virus. The initial
experiments were designed to determine the efficacy of this
procedure using the poliovirus type-1 strain CHAT, the strain
used for all experiments unless stated otherwise.
After mixing virus with sludge for 15 minutes at room
temperature and processing with SDS-sonication, there was
typically a 50 percent loss of recoverable infectivity from
the complete sample and an additional 17 percent loss from
the supernatant fraction, as shown in Table VI. As also
noted, the same treatment in phosphate buffered saline (PBS)
alone caused no loss of recoverable infectivity. This result
indicates that a portion of recoverable infectious virus
becomes bound to sludge solids and is removed with the pellet
during centrifugation. It also suggests that about 50 percent
of the virus is either rapidly inactivated in digested sludge
or has its infectivity masked by a component of sludge.
In order to determine whether the initial 50 percent loss
of poliovirus infectivity was due solely to trapping by sludge
solids, the effect of digested sludge on the recovery of
purified poliovirus labeled with 3H-uridine was examined. If
trapping were the only factor, then only about 33 percent of
the radioactivity should be recoverable in the supernatant
71
-------
to
TABLE VI
Recovery of Plaque-forming Units after
Mixing Poliovirus Strain CHAT with Digested
Sludge for 15 Minutes at Room Temperature
Sample/Treatment
Recovery of PFU
No sludge (PBS)/No treatment
No sludge (PBS)/SDS-sonication
No sludge (PBS)/SDS-sonication-centrifugation
Sludge/SDS-sonication
Sludge/SDS-sonication-centrifugation
2.4 x 10
8
2.4 x 108 (100)a
2.4 x 108 (100)
1.2 x 108 (50)
0.8 x 108 (33)
All numbers in parentheses are percentage recoveries relative
to the untreated control.
-------
after sedimentation of sludge solids. For this experiment,
labeled virus was mixed with sludge for 15 minutes and
processed with SDS-sonication. The samples were then centri-
fuged and the pellet was suspended in buffer, processed by
SDS-sonication and repelleted by centrifugation. After
repeating this procedure, the recovery of radioactivity and
plaque-forming units in each of the three supernatant frac-
tions was determined. As shown in Table VII, 64 percent
rather than 33 percent of the labeled virus was recovered in
the initial supernatant fraction. Further extraction of the
sludge solids resulted in the release of almost all labeled
virus. It is important to note that, if the number of plaque-
forming units obtained from total sludge is taken to be 100
percent recovery, then the percentage of radioactive virus
recovered during reextraction was parallel to the percent
release of viral plaque-forming units. This result strongly
suggests that the loss of infectivity upon addition of sludge
is due to viral inactivation. Moreover, viruses that remain
infectious after a short time in digested sludge are fully
recoverable by the techniques used here.
To further substantiate the conclusion that polioviruses
that retain their infectivities after mixing with digested
sludge are fully recoverable, other strains of the virus were
examined using these same procedures. As shown in Table VIII,
plaque-forming units of the type-1 strain Mahoney were re-
covered in toto from the complete sludge sample. However, as
with strain CHAT, a large portion of these infectious parti-
cles were bound to sludge solids because they were removed by
centrifugation. A similar result was obtained using the
type-2 strain 712. From this it seems clear that poliovirus
mixed with digested sludge becomes rapidly bound to solids;
but this association, at least initially, does not inhibit
the plaque-forming ability of the virus.
73
-------
TABLE VII
Recovery of Poliovirus in Digested Sludge
Supernatant upon Reextraction of Sludge Solids
Sample3
Total sludge
Sludge supernatant #1
Sludge supernatant #2
Sludge supernatant #3
Recovery of
Radioactivity (CPM)
11,070
7069 (63.9)b
2161 (19.5)
1139 (10.3)
(93.7)
Recovery of PFU
1.2 x 108
8.0 x 107 (66.7)
2.5 x 107 (20.8)
8.8 x 106 (7.3)
(94.8)
a 3
H-uridine-labeled poliovirus strain CHAT was mixed with digested
sludge for 15 minutes, processed with SDS-sonication, and analyzed
as described in the text.
Numbers in parentheses are percentage recoveries relative to CPM
present in total sludge or PFU recovered from total sludge.
-------
TABLE VIII
Recovery of Poliovirus (Strains Mahoney and 712)
after 15 Minutes in Digested Sludge
Sample
Recovery of PFUC
Strain Mahoney
Strain 712
No sludge (PBS)
Total sludge
Sludge supernatant
9.1 x 108
9.5 x 108 (104)b
3.2 x 108 ( 35)
1.8 x 10°
1.8 x 108 (100)
6.8 x 107 ( 38)
Average values from 3 duplicate experiments.
Numbers in parentheses are percentage recoveries relative to
sample without sludge.
Ul
-------
Loss of Infectivity of Poliovirus in Digested Sludge—
Anaerobic digestion of sludge typically occurs at temperatures
greater than ambient. For instance, the digesters at the
Albuquerque Treatment Plant are kept at about 35° C. At
this temperature poliovirus may be subject to rather rapid
heat inactivation. To minimize the inactivation due to heat
alone, the effect of digested sludge on poliovirus was studied
during incubation at temperatures between 28° C and 4° C.
Incubation in the absence of sludge caused the titer of
all three strains of poliovirus to decrease about one order
of magnitude during 5 days at 28° C, but little or no loss
was detectable during 3 days at 20° C or 5 days at 4° C, as
shown in Table IX. The presence of sludge at these temper-
atures caused the apparent rate of viral inactivation to be
greatly accelerated. This rate was greater than 1-log per
day at 28° C and about 1-log every 5 days at 4° C.
One implication of these results is that anaerobically
digested sludge contains a virucidal component whose activity
is temperature dependent. However, it is also possible that
the observed loss of titer was due to a time- and temperature-
dependent masking of viral infectivity by a component of
sludge. This possibility can be investigated by determining
the status of virus particles following incubation in sludge.
Although the properties of viruses in solution can be studied,
virus particles physically associated with sludge solids
cannot be readily analyzed. Therefore, in order to determine
whether the infectivity of poliovirus is irreversibly lost or
just masked in sludge it must be shown that virus that
remained bound to solids had been subject to the same effects
as virus found in the supernatant fraction following cen-
trifugation of solids.
76
-------
TABLE IX
Recovery of Poliovirus Plaque-forming Units from
Digested Sludge as a Function of the Time of Incubation
Incubation
Time/Temperature
1 day/280 C
3 days/200 C
5 days/40 C
5 days/280 C
Strain
CHAT
MAH
712
CHAT
MAH
712
CHAT
MAH
712
CHAT
MAH
712
Percentage Recovery
of PFU
- Sludge
69
55
70
83
82
100
100
100
100
6.3
11.0
8.4
+ Sludge
5.8
7.3
1.5
0.65
0.21
0.24
3.8
3.6
3.3
0.0003
0.00005
0.0002
77
-------
If bound and free virus is subject to the same effects
while in sludge, release of bound virus upon reextraction of
sludge solids should result in a proportional release of
piague-forming units. To measure the release of poliovirus
particles, virus labeled with H-uridine was incubated at
20° C for 3 days in digested sludge and processed by SDS-
sonication treatment. The sample was then centrifuged to
remove solids and the pellet was reextracted two additional
times by the same procedure. Finally, the radioactivity and
plaque-forming units recoverable in the three supernatant
fractions were measured. As shown in Table X, labeled virus
was almost completely recovered after three extractions of
sludge solids and the percentage release of radioactivity
during each extraction step was directly proportional to the
release of infectious virus. From these results it is con-
cluded that bound and free virus are subject to the same
effects during incubation in sludge. Therefore, radioactive
viruses found in the supernatant fraction following the first
centrifugation of sludge solids can be considered represent-
ative of all virus particles. The nature of these particles
was then studied to determine the mechanism by which polio-
virus loses its infectivity during incubation in digested
sludge.
Sedimentation Coefficient of Poliovirus after Incubation
in Digested Sludge—The possibility that a component of sludge
masks the infectivity of otherwise infectious poliovirus
particles seemed not unlikely in view of the number of papers
concerning substances that have this very effect. For ex-
ample, the methylthiopyrimidine S-7 has been found to bind
reversibly to poliovirus and temporarily block its infec-
19
tivity. For this reason, poliovirus was examined for
structural modification occurring during incubation in sludge
which would account for the observed loss of infectivity.
78
-------
TABLE X
Recovery of Poliovirus from Digested Sludge after Incubation
for 3 Days at 20° C by Reextraction of Sludge Solids
Sample
Recovery of
Radioactivity (CPM)
Recovery of PFU
Total sludge
Sludge supernatant #1
Sludge supernatant #2
Sludge supernatant #3
12,204
7,669 (62.8)'
2,632 (21.6)
1,302 (10.7)
(95.1)
1.3 x 10
6
8.0 x 10^ (61.5)
2.4 x 105 (18.5)
1.4 x 105 (10.8)
(90.8)
a Numbers in parentheses are percentage recoveries relative to CPM
present in total sludge or PFU recovered from total sludge.
-------
The initial effect on poliovirus during heat inactivation
has been reported to be the release of a protein called
VP-4. ' This loss causes the sedimentation coefficient of
the virus to decrease from 156s to about 130s. Further heat
treatment results in additional decomposition and concomi-
tant reduction in the sedimentation value of the viral
components. Although it has also been reported that both the
protein and RNA of poliovirus can be inactivated without
altering the sedimentation value of the particle,21'22 an
attempt was made to identify structural modifications through
changes in the sedimentation coefficient of the virus.
To measure changes in viral sedimentation rate, purified
poliovirus labeled with 14C-amino acids was incubated in
sludge and analyzed by centrifugation in glycerol gradients.
As shown in Fig. 32, incubation at 28° C both in the absence
and presence of sludge resulted in a considerable decrease in
the sedimentation rate of a portion of the labeled virus.
However, the percentage of particles detectably affected at
this temperature was significantly lower than the percentage
loss of infectivity (see Table IV). This is especially
evident for the sample containing sludge. Here, survival of
plague-forming units was less than 0.0003 percent while the
sedimentation value of only about 90 percent of the particles
had been visibly altered.
Loss of titer without a change in sedimentation coef-
ficient of a proportional fraction of virus was even more
evident after incubation at 4° C, as also shown in Fig. 32.
Although less than 4 percent of the original infectivity was
recoverable (see Table IV), there was almost no shift of
radioactive particles from the position of infectious virus.
These results show that if the loss of infectivity of polio-
virus in sludge is due to structural damage of the virion,
this damage is not necessarily reflected in the sedimentation
80
-------
3000 -
2000
-2000
-1000
•o-o-o-o-o-o-o-o-o-o-o-"
8 12 16 20 24
FRACTION NUMBER
Figure 32.
Effect of Sludge on the Sedimentation Coefficient
of Poliovirus. (The dashed line designates the
position of infectious virus.)
81
-------
values of the affected particles. Therefore, the individual
components of potentially inactivated particles having un-
altered sedimentation values were examined for structural
modifications.
RNA Component of Potentially Inactivated Poliovirus
Particles—The poliovirus particle is composed of a single-
stranded RNA genome and a capsid of 4 distinct proteins.
Because the genome is itself infectious, loss of viral
infectivity due to its damage will result in a proportional
loss of infectious RNA. Therefore, viral RNA was the first
component to be examined for structural damage.
Because breakdown of poliovirus is so much slower than
loss of infectivity in sludge at 4° C, alterations of viral
RNA were studied after incubation at this temperature. For
this, H-uridine-labeled virus was incubated for 10 days in
sludge during which time its recoverable infectivity decreased
99.1 percent. The sample was then processed by SDS-sonication
treatment in parallel with labeled virus which had been in
sludge at room temperature only 15 minutes. Even though some
loss of poliovirus infectivity takes place during a short
time in sludge (see Table VI) , this particular control was
included to ensure that any observed effect on viral RNA was
not caused by some sludge component present during processing
of virus or extraction of RNA. After pelleting sludge solids,
the samples were analyzed by density gradient centrifugation.
As shown in Fig. 33, the majority of particles in both
samples still sedimented at the same rate as infectious
virus (see Fig. 32). The peak fractions were combined as
shown and their RNA was extracted with phenol. The sedi-
mentation coefficients and infectivities of extracted RNA
were then measured.
82
-------
500
400
15 MIN,, R,T,
-------
Changes in sedimentation coefficient were determined by
density gradient centrifugation. As shown in Fig. 34, RNA
from the 15-minute sample was mostly intact, having a sedi-
mentation value of 35s as determined by co-sedimentation with
RNA extracted directly from purified virus. On the other
hand, RNA from virus in sludge for 10 days appeared to have
been nicked, having an average sedimentation value of less
than 25s. If incubation in sludge caused nicking of polio-
virus RNA, its specific infectivity should be considerably
less than that of RNA from the control sample. As shown in
Table XI, this is indeed the case. Furthermore, the ratio of
specific infectivities of virus/RNA for the 15-minute sample
was essentially identical to that of the sample in sludge for
10 days. From this it is concluded that loss of viral in-
fectivity parallels the decrease in infectious RNA. Thus,
the inactivation of poliovirus during 10 days in sludge at
4° C may be due to nicking of the viral genome within the
virus particle.
Because an intact, infectious RNA genome is a prereq-
uisite for an infectious poliovirus particle, these data
clearly establish one point. Viral plaque-forming units
were lost during these experiments as a result of irreversible
inactivation.
Proteins of Inactivated Poliovirus—Although the inac-
tivation of poliovirus RNA has been shown to occur in
particles whose sedimentation values had not been visibly
altered, the cause of inactivation has not been determined.
It is possible that ribonuclease present in sludge may
somehow penetrate the particle and produce limited digestion
of the viral RNA. However, incubation of poliovirus for
10 days at 4° C in 20 yg of ribonuclease per milliliter of
PBS at the same pH as sludge (pH 8.0) had no effect on its
infectivity. This result suggests that if ribonuclease is
84
-------
400
300
200
100
0
1 1
.
- 1
1
•
,«J
—
D
n-D^0
-TT-rT^O^TMT^n^g^r
1 1 1 1
iw
15 MIN., R.T.
\
sw
*^**»-.v ^.-.
D
/ \ 10 DAYS, 4°C
D-a a
/ \
a-a \j
/ \
\ -
a
n
^
-J 1 1 »
0
8
12
300
200
100
0
Q_
I
16
20
24
FRACTION NUMBER
Figure 34. Density Gradient Centrifugation of Poliovirus
RNA from Particles Recovered from Sludge.
(The dashed line shows the position of labeled
RNA extracted from untreated poliovirus and
sedimented in an identical gradient.)
85
-------
00
a\
TABLE XI
Infectivity of Poliovirus RNA Extracted
from Particles Incubated in Digested Sludge
Sludge Treatment
15 min. , 20° C
10 days, 4° C
Specific Infectivity3
(A) Virus
3.5 x 103
7.7 x 101
(B) RNA
1.9 x 10°
5.6 x 10"2
Ratio (A/B)
1.8 x 103
1.4 x 103
a Plaque-forming units per cpm of H-uridine.
-------
involved, some additional alteration of the particle probably
occurs which allows its penetration. The most likely alter-
ation is one involving the capsid protein of the virus. It
is improbable, however, that this alteration is of major
proportion since the sedimentation values of most particles
inactivated in sludge at 4° C were not detectably changed.
Conceivably, limited breakdown of capsid subunits may allow
penetration of the particles.
14
Possible breakdown of C-labeled poliovirus proteins
occurring during 10 days in sludge at 4° C was determined by
electrophoresis of inactivated, 156s particles in SDS-
polyacrylamide gels. The two largest viral proteins (VP-1
and VP-2) appear to have been broken down into smaller
peptides during incubation of virus in sludge, as shown in
Fig. 35. Based on the recovery of radioactivity in the
peak fractions of these gels versus total recovery, at least
75 percent of VP-1 and 25 percent of VP-2 were cleaved.
The upper limit of breakdown cannot be determined since
labeled peptides may have been released from the particles
during treatment or preparation of the samples for analysis.
It is of interest to note that VP-4, the first protein to be
released during heat inactivation of poliovirus, appears to
remain particle-associated. This cannot be definitively
stated, however, since some cleavage products of the large
viral proteins had molecular weights similar to that of VP-4,
but the finding that most particles inactivated at 4° C did
not have detectably altered sedimentation values supports
this suggestion. Therefore, the mechanism of heat inacti-
vation of poliovirus appears to be distinctly different from
the process occurring in digested sludge at low temperatures.
The results presented until now suggest that inactivation
of poliovirus in sludge at 4° C may be caused by the combined
effect of limited proteolytic digestion of viral particles
87
-------
0 10
20
30 HQ 50
FRACTION NUMBER
60 70 80
Figure 35. SDS-polyacrylamide Gel Electrophoresis
Pattern of Poliovirus Proteins from
Particles Recovered from Sludge.
88
-------
followed by penetration and inactivation of RNA by ribonuclease
Although it has been reported that incubation at 37° C in high
concentrations of various oroteases does not alter the infec-
tivity of poliovirus*23 the combined effect of proteolytic
enzymes and ribonuclease was studied under conditions simu-
lating those of this sludge experiment. As shown in Table XII,
essentially no reduction in the infectivity of virus was found.
Therefore, inactivation of poliovirus in sludge appears to be
caused by more than the combined effect of their two enzymatic
activities.
Absence of Virucidal Component in Raw Sludge—No consid-
eration has been given, up to this point, as to the origin of
the virucidal component found in anaerobically digested sludge.
This material may be a product of the digestion process or may
already be in the sludge prior to digestion. The latter
alternative seems unlikely in view of the finding that an-
aerobically digested sludge from two other cities (Los Angeles
and Denver) was found to have a similar amount of virucidal
activity to that of digested Albuquerque sludge (results not
shown).
In an attempt to determine the point of origin of the
virucidal material, raw sludge obtained in route to the
digester was measured for this activity. As shown in Table
XIII, raw sludge had no detectable effect on poliovirus
during 5 days at 20° C. This finding indicates that the
component of sludge responsible for inactivating poliovirus
originates in the digester.
Location of Sludge Component Responsible for Viral
Inactivation; Solids versus Liquid—In an initial step
toward the eventual identification of the components respon-
sible for viral inactivation, the liquid and solid portions
of digested sludge were separated and individually tested
89
-------
TABLE XII
Effect of Proteolytic Enzymes and
Ribonuclease on Poliovirus Infectivitya
Enzyme Treatment
None (PBS)
Trypsin
+ Ribonuclease
Chymotrypsin
+ Ribonuclease
Pronase
+ Ribonuclease
Recovery of PFU
2.0 x 10
8
2.0 x 10
8
1.8 x 10
1.8 x 10
8
Viral plaque-forming units were measured
following incubation with the given
enzymes for 10 days at 4° C and at the
pH found for digested sludge (8.0).
All protease concentrations were
100 yg/ml while that of ribonuclease
was 20 yg/ml.
90
-------
TABLE XIII
Effect of Raw Sludge on Poliovirus Infectivity
Sample
Recovery of PFU
Control
PBS (5 days, 20° C)
Digested sludge (5 days, 20° C)
Raw sludge (5 days, 20° C)
1.5 x 10
1.6 x 10
4 x 10'
1.4 x 10
8
-------
for their ability bo inactivate poliovirus. For this experi-
ment, sludge was centrifuged for 20 minutes at 18,000 g and
the supernatant decanted. The solids were then resuspended
in a volume of PBS equal to that of the removed liquid and
the two components were compared with the original sludge in
their abilities to cause poliovirus inactivation during 3 days
at 28° C. As shown in Table XIV, the material responsible
for loss of infectivity was found mainly in the liquid frac-
tion of sludge. Further analysis of this material is in
progress.
Heat Inactivation of Poliovirus in Raw and Anaerobicallv
Digested Sludge~~
A possible method of rapidly ridding sludge of viral
pathogens is with heat treatment. However, heating sludge
at high temperatures for extended periods of time is not
only a costly procedure, but one which may destroy a large
portion of its potential value. Therefore, if viruses are
to be inactivated in sludge by an elevation of temperature,
it is highly desirable to define an effective treatment
which requires a minimal amount of heat.
Viral disinfection in wastewater is commonly studied
using poliovirus as an indicator. A number of investigations
have been made concerning the rate and mechanism of inacti-
vation of this virus at various temperatures in defined
19 20 24—28
media. ' ' However, the rate of heat inactivation of
poliovirus in sludge has not been measured. Because a
variety of substances protect poliovirus against heat inacti-
vation (c.f. 19,29-31), the presence of sludge may cause a
considerable reduction in its inactivation rate. Therefore,
a study was undertaken to determine the effects of raw and
anaerobically digested sludge on the rate of heat inacti-
vation of poliovirus.
92
-------
TABLE XIV
Inactivation of Poliovirus by Various Fractions
of Digested Sludge During 3 Days at 28° Ca
Sludge Fraction
Recovery of PFU
Control
No sludge (PBS)
Total sludge
Sludge supernatant
Sludge solids
2.0 x 108
7.0 x 107 (35)b
5.7 x 103 (0.003)
6 x 102 (0.0003)
1.5 x 107(7.5)
Virus was incubated in various digested
sludge fractions following centrifugation
at 18,000 g for 20 minutes.
Percentage recovery relative to control
sample which remained frozen during the
time of incubation.
93
-------
Heat Induced Loss of Poliovirus Plague-Forming Units in
Sludge—The effect of raw and anaerobically digested sludge on
the rate of loss of poliovirus (strain CHAT) plaque-forming
units as a function of temperature was investigated. As shown
in Fig. 36, raw sludge is quite protective of the virus at all
temperatures studied here. On the other hand, the rate of
loss of plaque-forming units in digested sludge relative to
that occurring in buffer alone is dependent upon the temper-
ature. At the lowest temperature studied (43° C) digested
sludge is somewhat protective, but at the highest temperature
used (51° C) digested sludge accelerates loss of titer.
Probably the most significant feature of the data presented
in Fig. 36 is the dramatically different effects of raw and
digested sludge in these experiments. The apparent expla-
nations for this difference are that raw sludge either contains
a protective substance that is lost upon digestion or acquires
an activity during digestion that accelerates the rate of
heat inactivation of poliovirus. However, a third explanation
cannot be overlooked. Digested sludge may contain a component
that is stimulated by heat to become strongly bound to polio-
virus and, in so doing, mask the plaque-forming ability of
otherwise infectious viruses. This latter possibility can be
ruled out if it can be shown that the loss of poliovirus titer
with heat in digested sludge is due to viral inactivation.
Therefore, the physical nature of virus particles following
heat treatment in digested sludge was investigated.
Breakdown of Poliovirus During Heat Treatment in Anaer-
obically Digested Sludge—The nature of poliovirus particles
after heat treatment in digested sludge was studied by the
use of purified, radioactively-labeled virus. For this,
digested sludge was seeded with labeled virus, heated at
43° C for 200 minutes, and prepared for analysis by sonication
in 0.1 percent SDS. This treatment was found to cause about
94
-------
10
TIME (MINUTES)
Figure 36.
Effect of Sludge on the Rate of Heat Inactivation of Poliovirus,
Symbols: Raw Sludge (O) ; Digested Sludge (•); PBS (•).
-------
a 3-log decrease in recoverable plaque-forming units (see
Fig. 36). After heating, sludge solids were removed from
the samples by centrifugation at 18,000 g for 20 minutes and
virus retained in the supernatant fraction was analyzed.
The initial experiment was designed to determine virus
recovery. By measurement of total radioactivity, as shown
in Table XV, it was found that some radioactive material
(15 percent) is removed with the solids when the virus is
14
labeled with C-protein hydrolysate. However, when the
particles are labeled with H-uridine, radioactivity is
recovered in full. The difference between the percentage
recovery of viral RNA and viral protein can be explained by
examination of the recovery of acid-precipitable radioactivity.
As shown in Table XV, very little of the labeled viral RNA
remains acid-precipitable following heat treatment of polio-
virus particles in digested sludge while almost one-half of
the recoverable viral protein is still large enough to be
precipitable with acid. This result indicates that poliovirus
particles are broken down during heat treatment and that their
RNA molecules are hydrolyzed and released into the medium.
However, viral proteins are less extensively degraded than
viral RNA and a small percentage of these protein molecules
apparently remains associated with sludge solids during
centrifugation.
The conclusion that breakdown of poliovirus particles
occurs in digested sludge during heat treatment was confirmed
by a second experiment. Here, the sedimentation coefficient
of radioactively-labeled virus was measured. As shown in
Fig. 37, labeled particles have much lower sedimentation
values after than before heat treatment. Thus, poliovirus
is broken down and irreversibly inactivated when heated in
digested sludge.
96
-------
TABLE XV
Recovery of Radioactively-Labeled Poliovirus Strain CHAT
in Digested Sludge Supernatant after Heat Treatment
Radioactive Label
Sample
Radioactivity Recovered (CPM)
Total
Acid-precipitable
14
C-protein hydrolysate
H-uridine
unheated control (PBS)
heated 200 min, 43° C
in digested sludge
unheated control (PBS)
heated 200 min, 43° C
in digested sludge
1,685
1,432 (85.0)'
7,145
7,287 (102)
1,602
703 (43.9)
5,182
606 (11.7)
Numbers in parentheses are percentage recoveries relative to unheated control.
-------
1250
1000
750
500
250
0
BEFORE
n
0
O'
AFTER
A
/ \
D
/
\
D
\
I
D
/
/
/
I
D
\ '
*/
D
w
\
8
12
16
20
FRACTION NUMBER
Figure 37.
Sedimentation Profiles of Radioactively-Labeled
Poliovirus Before and After Heat Treatment in
Anaerobically Digested Sludge.
98
-------
1
Effects of Different Concentrations of Sludge on Heat
Inactivation of Poliovirus—In the experiments presented
above, undiluted sludge was seeded with poliovirus before
heat treatment. In an attempt to determine the difference
between the effects of raw and anaerobically digested sludge
seen in these experiments, the rate of heat inactivation of
poliovirus was studied after seeding lower concentrations of
sludge. The first experiment was designed to quantitate the
inactivation of strain CHAT during 200 minutes at 43° C over
a wide range of concentrations of digested sludge. The results
were quite unexpected. As shown in Fig. 38, extremely small
amounts of sludge are highly protective of the virus. However,
this protection diminishes as the concentration of sludge is
increased. At the highest concentration used, digested
sludge is almost as unprotective as PBS.
To examine the effect of sludge concentration in greater
detail, heat studies were carried out with all 3 strains of
poliovirus in low and high concentrations of both raw and
digested sludge. As shown in Table XVI, raw sludge is quite
protective at both concentrations but this capability is
especially evident in the greatest concentration at the
highest temperatures. Digested sludge, on the other hand,
is significantly protective only at relatively low temper-
atures and concentrations.
These results indicate that the protective component of
raw sludge is quite active even when present in very low
concentrations. Because the same low concentrations of
digested and raw sludge are almost equally protective at
the lowest temperatures studied, this component of raw sludge
appears to be retained after digestion. At higher sludge
concentrations and temperatures, the expression of the
protective component seems to be limited by another substance
found only in digested sludge. Thus, the difference between
99
-------
fe ur1
SLUDGE CONCENTRATION
(ML/2ML SAMPLE VOLUME)
Figure 38. Survival of Poliovirus After Heat
Treatment in Various Concentrations
of Anaerobically Digested Sludge.
100
-------
TABLE XVI
Effect of Sludge Concentration on Heat Inactivation on Poliovirus
Sample3
No sludge
0.05 ml raw sludge
1.8 ml raw sludge
0.05 ml digested sludge
1.8 ml digested sludge
No sludge
0.05 ml raw sludge
1.8 ml raw sludge
0.05 ml digested sludge
1.8 ml digested sludge
No sludge
0.05 ml raw sludge
1.8 ml raw sludge
0.05 ml digested sludge
1.8 ml digested sludge
No sludge
0.05 ml raw sludge
1.8 ml raw sludge
0.05 ml digested sludge
1.8 ml digested sludge
Treatment
39°, 200 min.
43° , 200 min
47° , 20 min.
51°, 5 min.
Percentage (%) Survival of PFU
Strain CHAT
3.4
95
98
69
4.3
0.088
53
72
5.8
0.056
0.024
6.4
64
0.013
0.0027
0.037
0.10
2.5
0.034
<0. 000026
Strain MAHONEY
67
93
59
64
4.7
1.5
74
67
39
0.093
0.43
60
60
4.1
0.0036
0.011
0.33
4.1
0.026
0.00028
Strain 712
77
83
62
83
28
0.26
98
74
29
5.3
0.066
108
100
5.0
0.18
0.023
23
52
0.038
0.0036
a
Each sample contained 0.2 ml poliovirus lysate, the specified volume of sludge and
the remainder as PBS in a total volume of 2.0 ml.
Survival was determined relative to unheated control in PBS.
-------
raw and digested sludge in these experiments may be due to a
virucidal activity acquired during digestion. The agent
responsible for this activity is possibly the same sludge
component previously shown to cause poliovirus inactivation
at much lower temperatures than those used here (see Part I
of this section).
Physical Separation of Sludge Components Affecting Heat
Inactivation of Poliovirus—Because anaerobically digested
sludge exhibits two activities having opposite effects on the
rate of heat inactivation of poliovirus, it should be possible
to physically separate the components responsible for these
activities. This was attempted by fractionation of sludge
into solids and liquid through centrifugation (18,000 g,
20 minutes) and comparison of the virucidal and protective
capacity of each to that of unfractionated sludge. As shown
in Table XVII, when the solids from a small amount of either
digested or raw sludge are resuspended in PBS, their protec-
tive capabilities are very similar to that of unfractionated
sludge containing an identical concentration of solids. In
contrast, an equivalent volume of the liquid fraction from
raw or digested sludge was found to be totally unprotective
in this experiment. These results show that the solids of
both raw and digested sludge contain most of the protective
component. They also support the previous conclusion that
raw sludge retains its protective capability after anaerobic
digestion.
The fraction of digested sludge that contains the
virucidal component was then determined. Because this agent
is much more readily expressed when present in high concen-
trations, heat inactivation of poliovirus was studied in
fractionated samples from undiluted sludge. As shown in
Table XVIII, most of the virucidal activity is removed with
the liquid portion of digested sludge. However, even when
102
-------
TABLE XVII
Survival of Poliovirus Strain CHAT after Heat Treatment in Fractionated Sludge
Sludge Concentration
No sludge
Lowa
Highb
Sample
PBS
total sludge
sludge supernatant
resuspended solids (PBS)
total sludge
sludge supernatant
resuspended solids (PBS)
(first centrifugation)
resuspended solids (PBS)
(second centrifugation)
resuspended solids (PBS)
(third centrifugation)
Percentage (%) Survival of PFU
after 200 Minutes at 43° C
Digested Sludge Raw Sludge
0.03
23 76
0.009 0.02
28 34
0.13 99
0.03 33
3.1 104
5.5 N.D.C
6.3 N.D.
Each sample contained either 0.05 ml of total sludge, the solids from this volume
of sludge, or an equal volume of digested sludge liquid in a total sample volume
of 2 ml.
Samples contained 1.8 ml of either total or fractionated sludge per 2 ml sample
volume.
Not determined.
-------
TABLE XVIII
Effect of Virucidal Activity on Heat Inactivation of Poliovirus in Raw Sludge
Sample
PBS
Digested sludge
Raw sludge
Raw sludge solids resuspended
in digested sludge supernatant
Percentage (%) Survival of PFU after
5 Minutes at 51° C
Strain CHAT
0.036
0.000034
1.1
0.0018
Strain MAHONEY
0.034
0.00023
3.7
0.00047
Strain 712
0.0055
0.00035
52
0.22
-------
the solids of digested sludge are washed several times with
PBS to remove this component, these solids are still not as
protective as those of raw sludge. In addition, a high
concentration of digested sludge solids is not as protective
as a low concentration. Therefore, it appears that a small
amount of virucidal activity is retained with this fraction
of digested sludge.
It should be noted that heat inactivation of poliovirus
under the conditions studied here is equally effective in PBS
and in undiluted supernatant from digested sludge. Because
this sludge fraction contains most of the virucidal agent, a
greater amount of inactivation was expected in it than in
PBS. The apparent explanation for this result is found upon
examination of the protective capability of a high concen-
tration of raw sludge supernatant. As shown in Table XVIII,
this quantity of raw sludge supernatant is highly protective.
Therefore, some protective material is retained in the liquid
fraction of raw sludge and is probably also still present
in the supernatant of digested sludge. From this it appears
that the actual rate of heat inactivation of poliovirus in
digested sludge supernatant, as in other fractions of digested
sludge, is determined by a competition between protective and
virucidal components.
Taken together, these results conclusively demonstrate
that anaerobically digested sludge contains both protective
and virucidal components and that these components can, for
the most part, be physically separated by the fractionation
of sludge solids and liquid through centrifugation.
Reversal of Raw Sludge Protection with the Virucidal
Agent of Digested Sludge—The solids of both raw and digested
sludge are highly protective of poliovirus during heat treat-
ment but inactivation occurs much more rapidly in digested
105
-------
than in raw sludge because a virucidal agent is acquired
during digestion. Because this agent is found primarily in
the liquid portion of digested sludge, it may be possible to
resuspend the solids of raw sludge in this liquid and reverse
the stabilizing effect on poliovirus normally provided by
these solids. This is indeed the case for all three strains
of poliovirus tested. As shown in Table XVIII, the amount of
heat inactivation of poliovirus that occurs during 5 minutes
at 51° C in raw sludge is much greater in the presence than
in the absence of the virucidal agent. In fact, the amount
of inactivation approaches that observed in digested sludge
under these conditions. Therefore, once this agent has been
identified, its addition to raw sludge should significantly
reduce the heat requirements needed to inactivate poliovirus
and possibly accelerate the inactivation of other viruses.
106
-------
1
PARASITOLOGY
Introduction
This project began in early FY 75. It had as its
objective, the determination of a reasonable treatment of
combined heat and radiation that would rid sewage sludge of
pathogenic parasites in an effort to make sewage sludge usable
as a soil conditioner or fertilizer on productive land.
The first stage in the project was the choice of a
parasite (and stage in its life cycle) to be studied. Some
of the criteria used in making this choice were that the
organism should be:
- a human pathogen
- found routinely in sewage sludge
- capable of surviving standard sludge digestion
procedures and environmental stress
- capable of study using in vitro methods (without
animals)
- more resistant to heat and radiation than other known
parasites satisfying the above criteria.
The final choice was the unembryonated ovum of Ascaris
lumbricoides. Ascaris ova are human as well as animal
pathogens. Ascaris is ubiquitous and its ova are found in
large quantities in sewage and tend to concentrate in sludge.
These ova are resistant to chlorination and heat and may
remain viable (and infective) for years in moist soil. In
107
-------
addition, of the parasite eggs found in sewage, those of
Ascaris species are most common. Finally, the embryonation
of Ascaris ova can be studied in vitro. In discussions with
recognized parasitologists, there was no disagreement with
the selection of Ascaris lumbricoides unembryonated ova as a
model system for studying the inactivation of sludge parasites.
The second phase of the project involved technique
development. While in vitro procedures for embryonating
Ascaris ova were available, methods of extraction of eggs from
worms, cleaning ova, preparing suspensions for treatment,
embryonating, and counting had not previously been developed
with the study of "survival" in mind. In addition, there
existed no single standard method for embryonation. Some of
the criteria for techniques were that they should:
- yield the highest number of ova after removal from
adult worms consistent with a low percentage of
unfertilized eggs
- minimize the loss of eggs to glassware during prepa-
ration of samples to be treated
- yield uniform environmental (heat and radiation)
exposure to all ova in a sample
- lend themselves to microscopic examination of ova
preparation at each stage
- yield maximum embryonation of untreated samples
- be as safe as possible
- yield needed information with minimal effort
Finally, concentration of the ova in sewage sludge was
required if inactivation measurements were to be made in
sludge with high (5 percent) solids content.
108
-------
Experimental
Preparation of Eggs
Ova were obtained from female Ascaris lumbricoides
measuring between 27 and 36 cm in length. These were ob-
tained from the Carolina Biological Supply Company. It was
determined that extraction of eggs from about 5 cm of the
uterus (posterior end) resulted in the maximal number of ova
with an acceptably small number of unfertilized eggs (6 to 8
percent). Also, from this portion of the uterus, the in
vitro embryonation procedures (see below) yielded about 93
percent embryonation of fertilized ova - complete to the
larval stage. The yield of fertilized ova per worm varied
from 6.1 x 104 to 5.5 x 105.
Uteri (5 cm) were deposited in IN NaOH, mashed into
small pieces with a glass rod, and stirred for 30 minutes
with a magnetic stirrer. These procedures were adopted to
aid in minimizing the loss of eggs through their adherence
to glassware. The use of a blender to fragment uteri caused
serious loss of ova.
The resultant mixture was poured through a 48-mesh
(295-ym) screen into a flask to remove uterine fragments.
After settling for 40 minutes, most of the NaOH was decanted
from the flask. Settling was used, rather than centrifugation,
to avoid exposure of ova to additional glassware (to which
they adhere). The ova settled readily (15 to 20 minutes) in
deionized water and 0.1N H2SO., but required 30 to 40 minutes
in NaOH. The ova were washed twice more in the same manner,
first with NaOH and then deionized water, after which
0.1N UjSO. was added to the cleaned suspension; this prepa-
ration was stored at 4° C.
109
-------
Other steps taken to minimize the loss of ova included:
the use of plastic disposable pipets whenever possible and
silicon-coating of most glassware - when not to be used with
NaOH which dissolves the silicon.
Sample Preparation
One milliliter of the stock suspension (above) was placed
in a 16 x 150 mm screw cap test tube. The suspension was
allowed to settle (30 minutes) and 0.5 ml liquid supernatant
was removed. Then 4.5 ml deionized water (or 3.5 ml water
and 1.0 ml sludge supernatant) was added.
Embryonation
Sample tubes (after exposure to various heat and/or
irradiation environments, or as controls) were decanted after
30 minutes settling, and 0.1N H2S04 was again added as the
in vitro embryonation medium. The ova were incubated for
21 days at 30° C on a slow roller drum. This choice of media
was made after considerable study, the results of which are
shown in Table XIX. (The numbers shown are lower than those
associated with the final procedures due to early inclusion
of a high percentage of unfertilized ova.) Generally,
I^SO. in air gave the best embryonation results. A hatching
technique was also developed, and it was found that all ova
which embryonated to the larval stage using the above pro-
cedure also hatched using this technique (the converse clearly
is true), so that any study of the life cycle beyond embryo-
nation to larval stage seemed unnecessary.
Counting
All counting for total number, number of fertilized eggs,
embryonated eggs, hatched larvae, and so forth was done by
suspending 0.1 ml of liquid (from appropriate state) in a
110
-------
TABLE XIX
Percent Embryonation in Selected Media
Concentrat ion
0.1N
0.1N
0.1N
2%
2%
2%
1%
1%
1%
Liquid
H2S04
H2S04
H2S04
HC1
HC1
HC1
Formalin
Formalin
Formalin
Gas
In CO2
In Air
In 02
In CO2
In O2
In Air
in CO2
in O2
in air
Percent
Embr yona t ion
Run I
53
51
49
47
42
36
52
47
35
Run II
56
43*
56
24
54
44
39
45
44
0.1N H2SO4 - Highest average,
- Least Deviation
-------
McMasters chamber and microscopically counting at 150X on a
Leitz microscope. Four counts were made for each sample and
means reported.
Microscopic counting is quite tedious. It was originally
planned to do most probable number counts and analyses for
hatching in this program in order to save time. This proved
to be impractical because there was too much variation in
initial counts necessitating microscopic examination in any
event.
Results
"Survival" (ability to embryonate to the larval stage)
curves for heat, radiation, and combination treatments were
generated by using the above techniques. Figure 39 shows
percent embryonation as a function of heat treatment at
several temperatures. These ova were exposed to heat in
4.5 ml DI water and 0.5N H2S04 suspension as described
earlier. Roughly, one can conclude that below about 51° C,
inactivation is slow; whereas above this temperature,
inactivation becomes increasingly rapid. Figure 40 shows
radiation and thermoradiation data (at 47° C) compared with
the total lack of effect of 47° C alone. In a very few
minutes, the combination of heat and radiation inactivates
three logs of embryonation synergistically.
Since there was an indication in some experimentation
that sludge supernatant afforded some "protection" against
the thermoradiation-induced inhibition of embryonation, it
became important to determine whether eggs in concentrated
sludge (5 percent) would be protected even more against such
a sewage sludge treatment process.
112
-------
O
C£
CO
10 -
10
10
30 40
TIME (MIN)
50 60
Figure 39. Heat Inactivation of Ascaris lumbricoides Ova
113
-------
<
ct:
ct:
00
IO'1 -
HEAT (47~ C for 2 hrs.)
RADIATION (20" C)
0
Figure 40.
80
DOSE (KRADS)
Thermoradiation Inactivation of Ascaris
lumbricoides Ova
114
-------
The techniques have been developed for separating Ascaris
lumbricoides ova from seeded sludge in order to visually
determine embryonation efficiencies using the formalin-ether
technique which has been reported.32'33 it has been deter-
mined in independent experiments that the formalin-ether
treatment does not interfere with subsequent embryonation of
the eggs.
Figure 41 shows the effects of ionizing radiation on the
embryonation of Ascaris lumbricoides ova in sludge and in
saline. While there appears to be some protection afforded
by the sludge, the difference is less than the normal batch-
to-batch variation in previously reported inactivation rate
studies.34 It is clear from these data, however, that
relatively "mild" treatment (150 krads) at 23° C will be
sufficient to prevent embryonation of three logs of parasite
ova; in addition, temperatures >45° C will yield even greater
success.
Summary
The inactivation data of Ascaris lumbricoides ova by
ionizing radiation for a variety of temperatures and in
various irradiation media are presented in Table XX. It is
clear that any radiation treatment, even at room temperature,
which includes as little as 150 krads of ionizing radiation,
will prevent embryonation in at least 99.9 percent of the
Ascaris ova in sewage sludge.
115
-------
20
40
DOSE(KRADS)
60
Figure 41. Reduction in Embryonation of Ascaris
lumbricoides Ova in Sewage Sludge
(5 Percent Solids) and Saline at 23° C.
116
-------
TABLE XX
Inactivation of Ascaris lumbricoides Ova
Irradiation
Temperature
(° C)
23
23
47
51
60
23
51
23
Inactivation
Medium
saline
water
water
water
water
supernatant
supernatant
sludge
Dose for
3 -Log Reduction3
(krads)
65b
85,140°
40
40d
10
140°
70d
90b
Reduction of embryonation ratio
b,c,d _ , .
Same dates
-------
EFFECTS OF HEAT AND IRRADIATION ON PHYSICAL/
CHEMICAL PROPERTIES OF SEWAGE SLUDGE
Introduction
Much of the cost of wastewater treatment in the United
States is directly related to the sludge handling problems
(greater than 30 - 40 percent by some estimates).35 Many
large plants presently dewater their sludge (to 20-25
percent solids), either by centrifugation or by vacuum
filtration. A large fraction of the dewatering cost (approxi-
mately 40 percent) is attributed to chemical additives
(ferric chloride, lime, alum or polyelectrolytes) which are
used for the purpose of facilitating a better separation of
the solids. There have been many reports in the last few
years on the improvements in dewatering properties brought
about by the use of ionizing radiation. If irradiation or
thermoradiation treatment can defray all or part of the
chemical costs, substantial savings can be realized as an
extra benefit (besides sterilization) to such a treatment
process. The goal of the research described in this section
has been quantification of radiation and thermoradiation
induced improvements in settling rates and in filtration.
Filterability measurements are somewhat more standardized,
and are more meaningful from an applications standpoint.
118
-------
Experimental
Settlability
Settling rates were measured by monitoring of solid-
liquid interface of 1-liter quantities of sludge in a
graduated cylinder. Figure 42 depicts the graduated cylinder
and the measurements taken. Improvement in settling is given
by
100 x
fc - LOO) treated - (Lfc - LQQ) control
- LOQ) control
or
100 ((AL) treated ,\
\ (AL) control L)
where the levels are as defined in the figure.
Filterability
The vacuum apparatus which was designed and built allows
measurement of the volume of filtrate as a function of time
for sludges which have been subjected to various treatment
conditions. This device is shown in Fig. 43. It consists
of a vacuum gauge, 500 millimeter vacuum flask, 12 millimeter
graduated cylinder, and a 9 centimeter Buchner funnel.
The following procedures were used in the filterability
experiments. A wetted piece of 7 centimeters Whatman Number 1
filter paper was placed in the Buchner funnel and a vacuum
was applied. A sludge sample of 50 millimeters was poured
into the Buchner funnel. The resulting vacuum was approxi-
mately 585 millimeters of mercury. The volume of the
119
-------
1000
, control
600>.
It, treated
400
Lt« liquid-solids interface
at time t
solids level at'Infinite11
time (depends on solids
content)
Figure 42. Apparatus for Settlability Measurements
120
-------
WHATMAN #1
FILTER PAPER
10 MILLILITER
GRADUATED
CYLINDER
BUCHNER FUNNEL
.FIXED VOLUME
^ OF SLUDGE
/TN
VACUUM
GAUGE
^^^r
TO VACUUM
Figure 43. Filtration Apparatus
121
-------
filtrate collected was recorded at 1/2 minute intervals for
the 1 percent solids sludge and at 1 minute intervals for the
3 and 5 percent solids sludge over a 10 minute period.
Results
Settlability
Only diluted (1 percent solids), digested sludge was
used. Prompt settlability was found to improve significantly
with radiation, to decrease slightly with heat, and to improve
significantly (and synergistically) with thermoradiation
treatment (Fig. 44). it should be noted that, while these
data indicate that at longer times the improvement is only
a few percent, this sludge was diluted to afford ease of
measurements. In typical, undiluted sludge, the improvement
may be long-term, or even permanent (days). Such has been
reported previously for irradiation alone.37 If this were
the case, an immediate improvement in sludge handling re-
quirements might be realized in a practical process.
Filterability
The data obtained from the filtration experiments were
plotted as time/unit volume versus volume. The slope, 'b,
of the line is related to the specific resistance of the
sludge. Specific resistance is defined as the resistance
of a unit weight of cake per unit area at a given pressure.
Specific resistance is expressed by the following equation:
= 2PA2b
yW
122
-------
o
O
1
60
TIME (MINUTES)
20 _
_L
40
TIME (MINUTES)
I
90
20 50
60
70
80
PERCENT SETTLED (CONTROL)
Figure 44.
Settlability Profiles for Heat, Radiation
and Thermoradiation
123
-------
where
r = Specific resistance, m/kg
P = Pressure difference, N/m2
A = Area of filtering surface, m2
b = Slope of the T/V vs. V plot, s/m6
y = Viscosity of filtrate, Ns/m2
W = Weight of dry sludge cake solids per unit volume
filtrate, kg/m3
Using a series of specific resistances obtained from the slopes
of the lines of the time/unit volume versus volume plots (Fig.
45, Plot A), a plot of normalized specific resistance versus
temperature or dose results (Fig. 45, Plot B). These plots
illustrate the change in specific resistance as a function of
treatment conditions. A decrease in specific resistance means
an increase in filterability.
Primary Digester Sewage Sludge
The results of the thermal treatment to 1, 3, and 5
percent solids sludge samples are shown in Fig. 46. In all
cases, heat alone increases the specific resistance of the
sludge, which results in increased difficulty in dewatering.
The detrimental effect increases in intensity as the per-
centage of solids increases. The 5 percent solids samples
exhibited approximately a 300 percent increase in specific
resistance while the lower percent solids samples exhibited
approximately a 25 percent increase in specific resistance.
The effects of radiation and thermoradiation on 5 percent
solids are shown in Fig. 47. Radiation alone decreases the
specific resistance greater than 5 fold at maximum treatment
124
-------
DOSE (MRADS)
3 1
1
^
2
1
3
1
4
1
o
0
2 3
VOLUME (MLS)
1.00
0.75 o
o
CO
£
o
0.50 5
LU
Q_
CO
O
uu
M
0.25
0
Figure 45. Radiation Effects on Filterability
A. Typical Time/Unit Volume versus
Volume Plot
B. Normalized Specific Resistance
versus Dose
125
-------
to
to
LU
oc
o
CL.
to
LU
IS1
o
Q
0.10
0.00
20.0
40.0 60.0 80.0
TEMPERATURE (°C)
100.0
Figure 46.
Thermal Effects on the Specific
Resistance of 1, 3, and 5 Percent
Solids—Digested.
126
-------
o
to
LU
oc
o
o
LU
Q_
to
O
LU
ISI
DC
o
o
o
-0.80
100.0
200.0 300.0
DOSE (Krads)
400.0 500.0
Figure 47. Radiation and Thermoradiation Effects
on the Specific Resistance of 5 Percent
Solids—Digested.
127
-------
conditions, while thermoradiation shows a 2 - 8 fold decrease
in specific resistance. Similar results were obtained for 1
and 3 percent solids.
For radiation the improvement is dose dependent, with
increased improvement up to 1000 krads, where a leveling off
occurs. The same trend is true for thermoradiation; however,
thermoradiation improvement is also dependent on temperature.
Thermoradiation at temperatures of 35 - 50° C show little or
no improvement over that at room temperature; however, a
significant improvement is observed at higher temperatures,
as seen in Table XXI.
The filterability of primary digester sewage sludge
increases half a log with radiation conditioning and as much
as a log with thermoradiation conditioning (depending on tem-
perature used). This seems to indicate that some agglomeration
of the solid material is occurring. This does not compare
favorably with the increases seen with chemical additives,
which typically increase filterability 2-3 logs.
It must be noted that for consistency all experimentation
was performed on strained and blended primary digester sludge.
The process of blending the sludge may have had a dramatic
effect on the filterability. For this reason studies on
fresh unblended sludge were initiated. Preliminary results
show that the blending did, in fact, reduce the filterability
of the primary digester sludge. It is apparent that the
blending process breaks up the agglomerations. This leads to
a decrease in filterability. The increase in filterability
of unblended primary digester sludge due to radiation or
thermoradiation is not, however, comparable to the increases
obtained by using chemical additives.
128
-------
TABLE XXI
Specific Resistance
m/kg) of 5 Percent Solids-Digested
Dose
(krads)
0
100
1000
21
1.67
0.968
0.444
Temperature (° C)
35
2.37
0.966
0.558
50
2.54
0.955
0.560
65
3.15
0.586
0.407
80
3.99
3.354
0.248
95
5.53
0.352
0.177
to
ID
-------
Undigested Sewage Sludge
Studies on the effects of heat, radiation and thermo-
radiation on the filterability of undigested sewage sludge
were undertaken. Initial results were very erratic. The
results are possibly due to the presence of activated sludge
which is being mixed into the undigested sludge at the sewage
plant. It is known that activated sludge is extremely
difficult to dewater. The experiments will be repeated on
unadulterated undigested sludge.
Summary
Significant enhancement of "prompt" improvement in
settlability has been measured for both irradiation and
thermoradiation treatment. Long-term effects have yet to
be demonstrated in our laboratory.
Filterability can be enhanced by a maximum of one log
by thermoradiation treatment in anaerobically digested
sludge which has been blended and strained. It is unknown
whether this improvement can defray some of the chemical
costs in dewatering operations, and tests are underway
currently to determine this. In addition, raw sludge and
non-blended anaerobically digested sludge are being studied
in terms of filterability improvement.
Odor modification studies are to be undertaken within
the next month. Quantitative data should be forthcoming
which will compare irradiation, heat, and thermoradiation
treatments of normal digested with normal raw sludge.
130
-------
COST BENEFIT ANALYSES
Cost benefit analyses for the treatment of sludge are
being carried out on a continuing basis as variables in the
thermoradiation process become better defined. A treatment
combination of 500 krads at 65° C for 5 minutes is used to
compute the cost of a rigorous treatment cycle. Based on
this treatment, analysis is presented below which should
yield approximate treatment costs based on current prices and
values. Gamma source efficiencies are presented for two
candidate source pins to be used in the thermoradiation
treatment process.
For a city of 600,000 population producing 91 g of
undigested sludge and 64 g of digested sludge per capita per
day, 760 M of 5 percent solids sludge is produced daily.
Also, 92.7 kcals/capita-day or an equivalent 644 kcal/second
total energy in the form of methane is available as output
from the anaerobic digestion process, which is currently
used in most sludge treatments. To provide the necessary
dose of 500 krad with a 35 percent source use efficiency,
137
32 MCi of Cs would be required at an approximate cost of
3.2 M$ (assuming a cost of 10
-------
a regular basis, we will assume that the total value of the
Cs is nominally the same at the end of a 20-year amortization
period as at the start, at least assuming rods at approximately
two-thirds the original specific activity are still useful. A
fairer way to amortize the source value will be derived later
and will increase the cost. Consequently, we end up with
0.75 M$ in construction cost amortized over a 20-year period
giving 37.5 K$/year. If the capital is amortized at 6 percent
the figure is 63.7 K$/year. Operating costs should be no more
than 100 K$/year and source decay amounts to 115.2 K$/year.
The total comes to 252.7 K$/year, while 14,000 tonne/year of
treated sewage sludge is produced. A cost of $18.15/tonne
or $20.00/tonne with 6 percent amortization is thereby ob-
tained for the assumed treatment.
The value of the treated sludge for fertilizer and for
the main ingredient in a cattle range feed supplement has
been discussed as thoroughly as the currently available data
permit with Dr. Stanley Smith and Dr. Robert McCaslin of
New Mexico State University in Las Cruces, New Mexico. Using
best estimates (chemical analysis is now being performed) of
the composition of Albuquerque sewage sludge, for example,
the treated sludge would be worth approximately $15/tonne
for fertilizer and as much as $100/tonne as animal feed.
Shipping costs and limited availability would restrict the
use of sludge at the cost of $18.15/tonne as a fertilizer
to the proximity of its source (less than 25 miles). Under
these circumstances, for example, the total sludge output of
Albuquerque would have limited impact on New Mexico's
fertilizer needs, but could furnish up to 25 percent of the
range feed supplement needs of the state.
As with any analysis of this type, gross generalizations
are involved. These generalizations are being examined more
closely. For example, roughly 50 percent of the $18.15/tonne
132
-------
137 137
cost is Cs cost and scales linearly with the cost of Cs.
Thus, a treatment involving only 250 krads (perhaps more
realistic) would decrease the cost of sludge per tonne by
about $4.50. More efficient ways of packaging the Cs are
being undertaken elsewhere at Sandia Laboratories. Since the
$.10/Ci value used in the analysis is almost totally one of
encapsulation costs, ways are being examined to simplify the
encapsulation procedure. The fertilizer and animal feed
value of the treated sludge are now being quantified accurately
by New Mexico State University.
Encapsula.tJ.on (EJEficl-ency Computations)
The major cost in building a full-scale sludge irradiation
137
facility is the cost of the source material ( Cs). Since
the presently available sources are relatively costly and
deliver gamma rays at somewhere around a 35 percent efficiency,
a study is being undertaken to quantify analytically the
radiation efficiency of various idealized source rod geometries.
All source geometries under consideration at this time
consist of fairly long rods (length >10 times the diameter) .
with cladding. The idealized geometry chosen to represent
this long but finite cylinder is an infinite cylinder with
cladding. Because only the radiation off the side of the
rod will probably be used and because the rods are long, this
idealized geometry should quite accurately predict the source
efficiencies. Computationally, because the point source
kernel rather than the line source kernel would have to be
used, the finite cylinder with cladding problem would have
been at least an order of magnitude more complicated and
time-consuming while providing very little added accuracy.
133
-------
The results of these computations are being used directly
in two cases. In the first case, the possibility of reducing
the diameter of the HMD Cs capsules from 6.35 cm to
approximately 2.54 cm is being discussed. This study will
quantify the gain in source efficiency. The second case is
the production of Cs in a ceramic matrix described above.
The rods being produced by this method are lower in specific
activity and higher in absorption because the cesium is mixed
with ceramic materials. However, because of the extremely
low-leach rate of the final product, the amount of cladding
needed can be reduced significantly. This study will quantify
in terms of source efficiency the various compositions and
geometries proposed.
The heart of the study is a computer program to evaluate
the photon flux at the surface of the rod. Since the surface
is convex, any photons that reach the surface escape and can
be used.
The source efficiency is defined to be total number of
photons that escape the rod divided by the total number of
photons released by the source material. Since all the y,b
and v2b are less than 1, the buildup factor is properly
neglected in these calculations. The geometry used is shown
below.
cladding
source
134
-------
The differential photon flux is given by
- y.^ [b sin 0 - Va2 - b2 cos2 0] + v^r 1
where
dA = 2irr dr d0
and
Ki(x) = / dx KQ(x)
where
KQ(x) - is a modified Bessel function of zeroth order
9
Da - disintegrations|cm - cm depth
V^ - absorption coefficient of the source material
V2 - absorption coefficient of the cladding
The final flux at the surface is given by
b sin 0 + Va2 - b2 cos2 0
* = 2 Da I I dr
J - sin"1 g b sin 0 - Va2 - b2 cos2 0
Ki
< (v2 - v^) |^b sin 0-Va2 - b2 cos2 0J + y^ | .
135
-------
Codes have not been written to evaluate the double integral
shown and to compute Ki(x). The final equation used is
Source efficiency = 2irby>
Da
The computer codes are now available in punched cards.
Dick Libby of Battelle PNL has recently found that build-
up factors increase the computed source efficiencies sub-
stantially (in some cases double). To resolve the differences
in computed efficiencies, a much more rigorous monte carlo
calculation will be performed.
Source Calculations
One approach suggested for increasing the source effi-
ciency of the WESF capsules, large diameter cesium-137
cylindrical sources being manufactured by ARHCO at Richland,
WA, was to leave a void in the center of the rod or to fill'
the center with a material with a low-absorption coefficient.
Using an infinite length stainless steel clad cesium chloride
rod with a cylindrical void in the center, source efficiency
calculations were undertaken. The geometry is shown below.
STAINLESS STEEL
CLADDING
136
-------
To compute the photon flux at the surface, it was necessary
to evaluate the following integrals:
r
0= 2Sa
/2"Sn e >-c sin 8 + \/b2 - c2 cos2 8
" / dr ___
- . m I y o n
i-sin - */c Sln 8 - Vb - c cos 8
2 c
Ki »c sin 8 +
d9 / dr
— la * ' A \/2
c sin 8 + \/b2 - c2 cos2 8
- c cos2 8
Ki
a2 - c2 Cos2 8
(r - c sin 8 - ^ , c2 cos
-------
Using this formula and defining source efficiency, the
following results are obtained where the dimensions are in
centimeters and the outer dimensions (b = 3.2 cm and
t = 0.508 cm), are similar to those of the WESF cesium
capsules.
Source Efficiency
Source Efficiency
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
26.8
26.0
25.4
24.9
24.5
24.2
24.1
24.0
1.8
2.0
2.2
2.4
2.6
2.8
3.0
24.1
24.3
24.6
25.0
25.7
26.5
27.5
As can easily be seen from the tabulation, it is of no
advantage to place the void in the center. The source effi-
ciency stays low until the thickness of the cesium chloride
layer is too small to be practical. This conclusion is in
agreement with Dick Libby of Battelle.
The cost/benefit analysis at this point is rather crude
in that so many assumptions must be made, such as, the price
of cesium-137, whether methane gas from the digester will be
available, etc. Further research has yielded answers to
many of these questions, and a final cost/benefit report will
be available in February 1977. An integral part of this
report will be the cost analysis developed by Battelle on
competing processes to thermoradiation, such as pasteurization
and irradiation. The Battelle results will be available in
December 1976.
138
-------
CONCLUSION
It has been demonstrated in the accumulation of the data
included in this report that irradiation, particularly at
elevated temperatures, is effective in destroying pathogens
and in improving sludge handling properties. Even at room
temperature the results are impressive. For example, a dose
of 300 krads will induce the following changes in digested
sludge:
(1) fecal streptococcus bacteria will be reduced by
about 2 logs (99 percent);
(2) coliform bacteria will be reduced by 10 logs;
(3) Salmonella species will be reduced by at least
8 logs;
(4) viable parasite ova will be reduced by 4 logs
at the very minimum;
(5) while viruses may be reduced only 1 log (90 percent),
by the irradiation, this work has demonstrated the
existence of a virucide for poliovirus, produced
in the process of digestion;
(6) settlability improves, and filterability is enhanced
by a factor of approximately 3.
139
-------
It must also be noted that increased temperature can
substantially enhance the expected inactivations, particularly
for viruses and parasite ova, and will lead to a further in-
crease in the filtering and settling properties.
Oxygenation applied during irradiation significantly
enhances inactivation rates of bacteria, even at elevated
temperatures. While virus inactivation may not be affected,
no data are yet available for parasite ova inactivation.
Cost analyses are currently under way to compare this
disinfection process with other processes, such as pasteur-
ization or more severe heat treatments.
FUTURE WORK
Experimentation to date has identified several areas in
which further study will help determine the value of the
proposed treatment. Further basic research in bacteriology,
virology, and parasitology is necessary to define these
parameters.
Bacteriological studies evaluating the effects of heat
on the pathogen Salmonella are intended to correlate with
our earlier work on radiation and thermoradiation inactivation.
Regrowth experimentation as well as competition experiments
affecting the regrowth may lead to the identification of
other factors concerned with bacterial inactivation in sludge.
The inactivation of biological systems by heat, radiation
and/or thermoradiation in undigested sludge is an area in
which there is a need for a whole series of experiments,
which we feel will benefit the total picture of sludge
characterization.
140
-------
I
Preliminary effects of oxygenation have been determined
for various bacteria. It is intended that this will be
combined with heat to give us more data as to the significance
of using oxygen in inactivation studies. The viability of
Ascaris ova after oxygenation has not yet been determined,
but will be correlated with bacteriological data. Aeration,
when compared to the oxygenation inactivation data, may well
be a feasible treatment from a cost effective viewpoint.
Ozonation in conjunction with radiation is another area in
which we will look at potential benefits.
Dried sludges which are then treated with irradiation
need to be analyzed for pathogen inactivation. In the event
that this becomes a more economically feasible treatment,
such information would be essential.
The agent of anaerobically digested sludge responsible
for inactivating poliovirus must be identified and its
activity against other viruses commonly associated with
sludge must be determined. Once it has been characterized
and its range of activity is known, it will then be important
to determine the real potential of the agent as a viral
inactivator during treatment plant operation. Because the
potential of this agent can be fully realized only if its
mode of action is understood at the molecular level, the
mechanism by which it inactivates viruses should be studied.
Finally, the possibility of extending its use to systems
other than sludge will be examined.
Biological modifications of anaerobic sludge digestion
by use of special strains of digester bacteria have been
considered. Proposals that deal with enhanced methane
production and conservation of fixed nitrogen by assimilating
denitrification are also being prepared. A lyophilized
culture of Chromobacterium violaceium has been obtained and
141
-------
will be used to study nitrate reduction to nitrite, and
nitrite to ammonia.
Odor modifications to be done in the very near future
include studies as follows:
(1) odor analyses on control samples of raw and of
digested sludge;
(2) analyses on samples heated to 40° C, 50° C, 70° C
and 95° C;
(3) analyses on irradiated samples (250, 500, 750
and 1000 krads);
(4) analyses on thermoradiation-treated sludges for
a variety of doses and temperatures.
These studies are being performed at Battelle PNL in
Richland, Washington.
Filterability studies are planned which will show if
the decrease in filterability induced by irradiation or
thermoradiation treatment can offset any chemical costs of
a standard dewatering process, such as vacuum filtration
or centrifugation.
The major aim for the cost benefit analysis is the
preparation of a final cost/benefit document, which will be
available in February 1977. The final assembly of the
document is awaiting cost results on competing processes
from studies under way at Battelle, PNL. The Battelle
results will be available in December 1976. One major set
of monte carlo computer calculations, that are needed to
corroborate gamma source efficiency calculations done by
142
-------
Dick Libby of Battelle, PNL, will be performed this year.
In addition, irradiator design calculations will be done
to estimate total source use efficiencies for the cost
benefit analysis.
143
-------
References
1. M. C. Reynolds, R. L. Hagengruber, and A. C. Zuppero,
Thermoradiation Treatment of Sewage Sludge Using Reactor
Waste Fission Products, SAND74-001, Sandia Laboratories.
June 1974.
2. R. Sullivan, et al, Applied Microbiology, 21^ 61, 1971.
3. R. Sullivan, et al, Applied Microbiology, 22_, 315, 1971.
4. R. Trujillo, Measurement of the Radiation Inactivation of
Attenuated Poliovirus by the Plague Assay Technigue,
Sandia Laboratories, Albuquerque, New Mexico, SLA-74-0065.
5. G. E. Milo, Applied Microbiology, 22, 198, 1971.
6. This work.
7. C. A. Lawrence and S. S. Block, Disinfection, Sterilization,
and Preservation, Lea and Febiger, Philadelphia, 750, 1968.
8. C. T. Thomas, et al, Applied Microbiology, 14, 815, 1966.
9. F. J. Ley, IAEA 73/35, 1973.
10. (a) This work. (b) G. Baney, Ph.D. Dissertation, Purdue
University, 1970.
11. "Standard Methods for the Examination of Water and
Wastewater," American Public Health Association, 1971.
12. Ibid, p. 691.
13. B. A. Kenner and H. P. Clark, "Detection and Enumeration
of Salmonella and Pseudomonas aeruginosa," J. Water
Pollution Control Fed., 46, 2163, 1974.
14. B. Kenner, G. K. Dotson, and J. E. Smith, "Simultaneous
Quantitation of Salmonella Species and Pseudomonas
aeruginosa," EPA, NERC, Cincinnati, OhicT
15. H. D. Sivinski, et al, Industrial Sterilization Inter-
national Symposium, Amsterdam, 1972, Duke University
Press 17, 1973.~
144
-------
16. J. R. Brandon and S. L. Langley, Inactivation of Bacteria
in Sewage Sludge by Ionizing Radiation/ Heat, and Thermo^
radiation, SAND75-0168, Sandia Laboratories, 1976.
17. E. Lund and V. Ronne, "On the Isolation of Virus from
Sewage Treatment Plant Sludges," Water Res., 7_, 863-871,
-L 7 / ,3 •
18. A. Palfi, "Survival of Enteroviruses During Anaerobic
Sludge Digestion," in Advances in Water Pollution Research,
S. H. Jenkins, ed., Proceedings of the Sixth International
Conference, Jerusalem. Permagon Press, New York, 99-104.
19. K. Lonberg-Holm, L. B. Gosser and J. C. Kauer, "Early
Alteration of Poliovirus in Infected Cells and its
Specific Inhibition," J. Gen. Virol., 27_, 329-342, 1975.
20. M. Breindl, "The Structure of Heated Poliovirus Particles,"
J. Gen. Virol., 1^, 147-156, 1971.
21. R. M. Franklin and E. Weeker, "Inactivation of Some
Animal Viruses by Hydroxylamine and the Structure of
Ribonucleic Acid," Nature, 184, 343-345, 1959.
22. B. Oberg, "Biochemical and Biological Characteristics of
Carbethoxylated Poliovirus and Viral RNA," Biochim.
Biophys. Acta., 204, 430-440, 1970.
23. D. 0. Cliver and J. E. Herrmann, "Proteolytic and
Microbial Inactivation of Enterviruses," Water Res., 6,
797-805. -
24. N. J. Dimmock, "Differences Between the Thermal Inacti-
vation of Picornaviruses at 'High1 and 'Low1 Temperatures,"
Virology, 31, 338-353, 1967.
25. V. L. Dugan and R. Trujillo, "Heat-accelerated Radio-
inactivation of Attenuated Poliovirus," Rad. Environ.
Biophys., 12_, 187-195, 1975.
26. Y. Hinuma, S. Katagiri, M. Fukuda, K. Fukushi, and
Y. Watanabe, "Kinetic Studies on the Thermal Degradation
of Purified Poliovirus," Biken J., 8_, 143-153, 1965.
27. G. Koch, "Influence of Assay Conditions on Infectivity
of Heated Poliovirus," Virology, 12, 601-603, 1960.
28. J. S. Youngner, "Thermal Inactivation Studies with
Different Strains of Poliovirus," J. Immunol., 78,
282-290, 1957. —
145
-------
29. P. Pohjanpelto, "Stabilization of Poliovirus by Cvstine "
Virology, 6, 472-487, 1958. y^me,
3°* -I ?:,?teele and F« L- Black, "Inactivation and Heat
Stabilization of Poliovirus by 2-Thiouracil " J
Virol., 1, 653-658.
31. C. Wallis and J. L. Melnick, "Cationic Stabilization—
A New Property of Enteroviruses," Virology, 16, 504-506,
32. L. S. Ritchie, "An Ether Sedimentation Technique for
Routine Stool Examinations," Bull. u. S. Army Med
Dept., 8_, 326, 1948.
33. M. L. Peterson, "Parasitological Examination of Compost,"
U. S. EPA Solid Waste Open-File Report, 1971.
34. J P. Brannen, D. N. Garst, and S. L. Langley, Inactivation
of Ascans Lumbricoides Eggs by Heat, Radiation and Thermo-
radiation, SAND75-0163,Sandia Laboratories1975'
35. H. Bernard, "Alternative Methods for Sludge Management,"
Municipal Sludge Management, Proc. of National Conf. on
Municipal Sludge Management, Information Transfer Inc
p. 11, 1974.
36• Process Design Manual for Sewage Treatment and Disposal
Environmental Protection Agency, October 1974.
37. T. Lessel, H. Motsch, E. Hennig, A. Suess, A. Rosopulo,
G. Schumann, "Experience With a Pilot Plant for the
Irradiation of Sewage Sludge," IAEA-SM-194/604, p. 447,
-L .7 / O o
146
-------
DISTRIBUTION:
George P. Dix
Associate Director
Division of Nuclear Research
and Applications
ERDA Headquarters
Mail Station A2-2200
Washington, DC 20545
Earl Walquist
Division of Nuclear Research
and Applications
ERDA Headquarters
Mail Station A2-2200
Washington, DC 20545
William Remini
Division of Nuclear Research
and Applications
ERDA Headquarters
Mail Station A2-2200
Washington, DC 20545
Gerald Stern (5)
Ultimate Disposal Research
Program
Environmental Protection
Agency - MERL
Cincinnati, OH 45268
J. B. Farrell
Ultimate Disposal Research
Program
Environmental Protection
Agency - MERL
Cincinnati, OH 45268
William A. Rosencranz
Waste Management Division
Office of Air, Land and
Water Use
Environmental Protection
Agency
Waterside Mall (RD-682)
Washington, DC 20460
Mildred Smith
Special Assistant for Research
and Development
Environmental Protection
Agency
1600 Patterson Street
Dallas, TX 75201
D. K. Nowlin
Attn: D. L. Krenz
Special Programs Division
ERDA/ALO
Albuquerque, NM 87115
G. Stanley Smith
Department of Animal, Range,
and Wildlife Sciences
New Mexico State University
Las Cruces, NM 88003
Bob McCaslin
Department of Agronomy
New Mexico State University
Las Cruces, NM 88003
John D. Purcell
N. M. State Rural Development
Committee
Farmers Home Administration
517 Gold, SW
Albuquerque, NM 87102
Erwin Hensch
Public Works Department
P.O. Box 1293
Albuquerque, NM 87103
Donald Tryk
Chief, Engineering Division
New Mexico Environmental
Improvement Agency
P.O. Box 2348
Santa Fe, NM 87503
Tom Baca
New Mexico Environmental
Improvement Agency
P.O. Box 2348
Santa Fe, NM 87503
Jerry Sargent
Division of Water and Air
Quality Control
Department of Environmental
Conservation
State of Alaska
Mail Pouch O
419 6th Street
Juneau, AK 99801
147
-------
DISTRIBUTION: (cont)
Fred Reiff, Director
Office of Environmental Health
U.S. Public Health Service
3550 Commercial Drive
Anchorage, AK 99510
Richard W. Latimer
Arctic Environmental Research
Laboratory
Environmental Protection
Agency
College, AK 99701
1140 W. D. Weart
1700 0. E. Jones
5000 A. Narath
Attn: J. K. Gait
E. H. Beckner
A. Y. Pope
J. H. Scott
5400 A. W. Snyder
5410 D. J. McCloskey
5420 J. V. Walker
5430 R. M. Jefferson
5440 H. D. Sivinski (30)
5441 C. A. Trauth, Jr.
5442 W. J. Whitfield
5443 R. E. Luna
5800 R. S. Claassen
5820 R. L. Schwoebel
5824 R. W. Lynch
3141 C. A. Pepmueller (5)
8266 E. A. Aas (2)
3151 W. L. Garner (3)
for ERDA/TIC (Unlimited
ERDA/TIC (25)
(R. P. Campbell, 3171-1)
THIRD DISTRIBUTION:
Gerald Stern (200)
Ultimate Disposal Research
Program
Environmental Protection
Agency - MERL
Cincinnati, OH 45268
William Remini (50)
Division of Nuclear Research
and Applications
ERDA Headquarters
Mail Station A2-2200
Washington, DC 20545
5445 H. D. Sivinski (50)
Release)
SECOND DISTRIBUTION:
5442 W. J. Whitfield (50)
148
------- |