Environmental Protection Technology Series
EVALUATION OF HEALTH HAZARDS
ASSOCIATED WITH SOLID WASTE/
SEWAGE SLUDGE MIXTURES
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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EPA-670/2-75-023
April 1975
EVALUATION OF HEALTH HAZARDS ASSOCIATED WITH
SOLID WASTE/SEWAGE SLUDGE MIXTURES
By
William L. Gaby
Department of Health Sciences
East Tennessee State University
Johnson City, Tennessee
Program Element No. 1DB064
Project Officers
Mirdza L. Peterson and Clarence A. demons
Solid and Hazardous Waste Research Laboratory
National Environmental Research Center
Cincinnati, Ohio 45268
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
For sale by the Superintendent of Documents, U.S. Government
Printing Office, Washington, D.C. 20402
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REVIEW NOTICE
The National Environmental Research Center -
Cincinnati has reviewed this report and approved its
publication. Approval does not signify that the contents
necessarily reflect the views and policies of the U.S.
Environmental Protection Agency, nor does mention of
trade names or commercial products constitute endorse-
ment or recommendation for use.
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FOREWORD
Man and his environment must be protected from the adverse
effects of pesticides, radiation, noise and other forms of pollution,
and the unwise management of solid waste. Efforts to protect the
environment require a focus that recognizes the interplay between
the components of our physical environmentair, water, and land.
The National Environmental Research Centers provide this multidisci-
plinary focus through programs engaged in
O studies on the effects of environmental contaminants on
man and the biosphere, and
O a search for ways to prevent contamination and to recycle
valuable resources.
This report summarizes and evaluates the health hazards associ-
ated with municipal solid wastesewage sludge composting by the
windrow composting process. It is concluded that a properly composted
solid waste or solid waste-sewage sludge mixture is microbiologically
acceptable for many uses without creating health hazards.
A. W. Breidenbach, Ph.D.
Director
National Environmental
Research Center, Cincinnati
111
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ABSTRACT
The composting of refuse-sewage sludge by the windrow pro-
cess results in the aerobic biodegradation of organic solids
and liquids to a relatively stable end product which may be
used as a soil conditioner without creating health hazards or
pollution of water, land or air. Composting is a rapid and
natural process by which all organic matter is decomposed by
microorganisms to inorganic compounds or elements which are
utilized by other plants and animals. This cyclic trans-
formation is an essential process without which all plant
and animal life would cease. Anaerobic biodegradation also
occurs in such processes as landfills but at a much slower
rate. The microbial ecology of compost is directly related
to the internal temperature of the windrow. These studies
indicate that large numbers of microorganism present in
refuse and sewage sludge utilize the nutrients available,
releasing excessive energy which increases the temperature
of the windrow to a maximum of approximately 167 F (74 C)
within 7 days. The disappearance of inserted selected patho-
genic microorganisms from compost is directly related"to
this temperature increase and not to any type of antagonistic
action resulting from antibiotic activity or other metabolic
products of microorganisms in compost. Proper processing,
such as aeration and moisture is required for the windrow to
reach a temperature of 120 F to 167 F (49 C to 74 C) or
greater for a period of 4 to 7 days. If the windrow tempera-
ture does not reach 120 F (or falls below 120 F), the micro-
bial flora and pathogens remain viable at a high level and
may increase in numbers.
Temperatures observed in the top and bottom 2-4 in layers of
the windrows were extremely variable and could not ensure the
destruction of pathogens unless the windrows were properly
turned.
The handling and disposal of refuse and refuse-sewage sludge
should be considered a health,hazard and a potential source
of many microbial infections.
IV
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CONTENTS
Page
ABSTRACT iv
LIST OF FIGURES vi
LIST OF TABLES vii
CONCLUSIONS 1
RECOMMENDATIONS 4
INTRODUCTION 5
Scope 5
MATERIALS AND METHODS 8
Sampling procedures 8
Microbiological procedures 10
Total and fecal coliforms 10
Coagulase positive staphylococci 11
Salmonella and Shigella 11
MPN count 12
Enteroviruses 12
Parasites 12
Pathogenic fungi 13
Insertion studies 14
Bacteria 14
Recovery of organisms from broth and agar slant cultures 15
Recovery of organisms from filter paper discs 15
Salmonella typhimurium 15
Shigella sonnei 16
Enteroviruses and Leptospira 16
Fungi 16
Parasites 17
Background studies 17
RESULTS 18
REFERENCES 45
v
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LIST OF FIGURES
FIGURE PAGE
1. Windrow cross sections and sampling pro-
cedures ................... ?
2. Average number of total coliforms, fecal
coliforms and fecal streptococci in raw re-
fuse, sewage sludge and refuse-sewage sludge
mixture on zero day
3. Average number of total coliforms, fecal
coliforms and fecal streptococcus in refuse-
sewage sludge on zero day for the four
seasons .................... 20
4. Average total coliform MPN count from re-
fuse-sewage sludge windrows at 2-4 in.
depth .................... 22
5. Average total coliform MPN count from re-
fuse-sewage sludge windrows at 6-8 in.
depth .................... 24
6. Average total coliform MPN count from re-
fuse-sewage sludge windrow at 2 ft depth - - - 25
7- Average total coliform MPN count from re-
fuse-sewage sludge windrow at 6-8 in. from
bottom ................... 26
8. Average total coliform MPN count from re-
fuse-sewage sludge windrow at 2 in. from
bottom ................... 27
9. Total plate count of raw refuse
windrow .................. . 28
10. Total plate count of refuse-sewage sludge
windrow
29
VI
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FIGURE PAGE
11. Survival time in windrow of total coli-
forms, fecal coliforms and fecal strepto-
cocci 30
12. Survival time in windrows of Bacillus sp.,
Chromogenic sp. and Pseudomonads 31
13. Survival time in windrow of Proteus and
coagulase positive staphylococci 33
14. Survival time in windrow of E. coli, S.
aureus, Sal, typhimurium and Sh. sonnei ... 35
15. Survival time in windrow of E. coli, J3.
aureus, Sal, typhimurium and Sh. sonnei . . . 3g
LIST OF TABLES
TABLES
1. Species of Salmonella isolated from raw
refuse, raw sewage sludge and refuse-
sewage sludge mixture on zero day 21
2. Proteus and coliforms in soil 34
3. Survival of microorganisms in refuse-sewage
sludge windrow 1-23-69 to 2-27-69 39
4. Survival of microorganisms inserted in re-
fuse-sewage sludge windrow 1-17-68 to 2-13-
68 40
5. Survival of microorganisms inserted in re-
fuse-sewage sludge windrow 10-21-68 to 11-12-
68 41
6. Survival of human parasites inserted in re-
fuse-sewage sludge windrow 42
7. Survival of Canine parasites inserted
in refuse-sewage sludge windrow 43
VII
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CONCLUSIONS
The end product of refuse-sewage sludge composting by the
windrow process results in a relatively stable product
comprised primarily of humus. These studies have shown
that a properly composted refuse or refuse-sludge mixture
can safely be used as a soil conditioner for gardens,
farms, and lawns or as an ideal material for filling gull-
ies and areas of erosion without creating public health
hazards. The disappearance of pathogenic microorganisms
from compost was found to be directly related to tempera-
ture. Proper composting, therefore was dependent upon
the windrow reaching a temperature of 120F-167F (49C-74C)
or greater for a period of at least 4 to 7 days. If for
any reason, i.e., extreme cold temperatures, improper
turning, or anaerobiosis, the windrow temperature does not
reach 120 F or falls below 120 F during the process the
bacterial flora, including the pathogens originally present
in refuse and sewage sludge remains at a high level or
even increases significantly in numbers.
The center or midpoint of the windrow reaches a consistently
high temperature of 120 F and greater, assuring the destruc-
tion of pathogens. However there is considerable varia-
tion in the temperature of the top and bottom 2 in. of
the windrow indicating that proper turning and mixing of
the compost is essential to ensure destruction of all
pathogens in windrow composting.
Species of Salmonella and Shigella are present in raw re-
fuse and sewage sludge in relatively small numbers. How-
ever these gram-negative, pathogenic enteric bacilli
originally present or inserted into the refuse-sewage
sludge windrows under controlled conditions, disappeared
from the windrow within 7 to 21 days.
Enteroviruses were not isolated from raw refuse, sewage
sludge or refuse-sludge compost. Type 2 poliovirus in-
serted into the windrows were inactivated after 3 to 7
days exposure to 120 F. It is evident from these results
that enteroviruses do not present a public health hazard
in the properly processed compost and that polioviruses
do not survive the composting environment.
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Intact parasitic ova could be observed throughout the com-
posting process. However the ova observed in the finished
product were not of human origin and would not limit the
use of compost. Human parasitic cysts and ova inserted in
the center of the windrow were disintegrated after 7 days
exposure. Dog parasitic ova remained intact 35 days after
insertion in windrows at 2 in. and mid-depth. Knowing
that pet excreta may be present in residential refuse the
survival of these intact ova should be further investigated.
However one would expect to find under normal conditions
significant numbers of animal parasitic ova in all subur-
ban areas in which there are pet dog and cat population.
The spirochate Leptospira Philadelphia was found to be
extremely sensitive to composting temperatures and did
not survive for more than 2 days when inserted in windrows.
Several genera of molds where observed in the refuse-
sewage sludge compost throughout the process and were
especially numerous during the latter stage. The common
occurring, easily recognized genera included Mucor, Rhizo-
pus, Penicillium, Aspergillus, Cladosporium and Cephalo-
tecium. The pathogenic fungi Blastomvces dermatitidis
and Histoplasma capsulatum were never isolated from raw
refuse-sewage sludge mixtures and insertion studies in-
dicated that these pathogenic fungi did not survive
composting temperatures. In only one instance did a
culture of Histoplasma capsulatum survive for as long as
26 days and this culture has been inserted in the top 2
to 4 in. of the windrow where there was considerable
temperature variation. Cultures of Asperqillus fumigatus
were readily killed when inserted in the windrows at
temperatures of 120 F of greater. However this mold is
normally present in refuse and could be isolated from the
compost throughout the process.
Insertion studies carried out by Morgan (18) revealed
that Mycobacterium tuberculosis (avirulent M. tuberculosis
var. hominis H37RA) was destroyed within 2 weeks by the
high temperatures attained in windrow composting.
Proteus species and in particular, Proteus mirabilis was
present in all refuse or raw refuse-sludge compost samples
examined. Furthermore, Proteus could consistently be
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isolated from soil in which refuse had been mixed. When
the refuse decomposed, Proteus could no longer be isolated.
These results suggest that Proteus may be an idea indicator
organism for the presence of raw or partially decomposed
refuse or compost.
The most significant finding of public health interest or
concern resulting from this study is that the public health
hazards resulting from the handling and disposal of raw
refuse are equivalent to or greater than those resulting
from the handling and disposal of raw sewage sludge.
These results indicate that the total microbial flora of
refuse is equal to that of raw sewage sludge. Not only does
refuse contain all of the microorganisms found in
raw sewage sludge but also all of the organisms found in
the upper respiratory tract of man (12). Therefore the
handling of and the exposure to raw refuse should be
considered a health hazard and a potential source of
numerous infections including impetigo, a contagious
disease caused by staphylococci and streptococci, diph-
theria, streptococcus sore throat, upper respiratory in-
fections including influenza and the common cold.
The disposal of raw solid waste is fundamentally a health
problem (1, 24). The results of this study clearly
indicate that disposal of raw refuse in open dumps or in
landfills constitutes as great a public health hazard as
would the dumping of raw dewatered sewage under the same
conditions. Covering raw refuse with soil creates an
anaerobic condition, comparable to that of an anaerobic
windrow, under which sufficient heat is not generated to
kill undesirable microorganisms and since most of the
pathogenic bacteria found in raw refuse are facultative
anaerobes these organisms would survive. Since landfill-
ing is the only method of solid waste disposal recommended
by the Environmental Protection Agency (3) the health
hazard associated with landfilling should be thoroughly
investigated. Such a study has never been made.
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RECOMMENDATIONS
1. The most significant finding of public health interest or
concern resulting from this study is that the public health
hazards resulting from the handling and disposal of raw
refuse are equivalent to or greater than those resulting
from the handling and disposal of raw sewage sludge. These
results indicate that the total microbial flora of refuse
is equal to that of raw sewage sludge.
2. Based on these studies aerobic windrow compost where
properly processed, does not constitute a public health
hazard and is an excellent soil conditioner for gardens,
lawns and other areas. Compost should not be limited in its
use any more than any other soil conditioner.
3. Use of material from anaerobic windrows would constitute
a public health problem since microorganisms are not de-
stroyed.
ADDITIONAL RESEARCH NEEDS
1. An educational program should be initiated to inform the
general public of health hazards associated with the handl-
ing of raw solid waste in the home and recommend types of
refuse disposal containers.
2. Additional studies should be made to confirm the results
indicated in this study of the possible thermo resistance
of dog parasitic ova.
3. Studies are needed to determine possible means of re-
ducing the cost of recycling solid wastes.
4. Research is needed to determine the possible use of 14
to 21 day old compost for various reclamation projects.
5. Reduce cost of recycling by homeowners separation of
biodegradable and nonbiodegradable wastes through local
ordinance.
6. Since the only method of solid waste disposal recommended
by the Environmental Protection Agency is landfilling, more
intensive studies should be made to determine the survival
of microorganisms in solid waste under these anaerobic
conditions.
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INTRODUCTION
This report summarizes and evaluates the health hazards
associated with solid waste sewage sludge composting from
data accumulated during the period from June 1, 1967 through
May 31, 1969 under contracts No.PH-86-67-112 and No.86-68-
143 between the U.S. Public .Health Service and East Tennessee
State University, Department of Health Sciences, College of
Health, Johnson City, Tennessee. The study was initiated as
a result of a cooperative joint project agreement between
the U.S. Public..Health Service, The Tennessee Valley Author-
ity and the Municipality of Johnson City. Tennessee (15).
The scope of work included in these contracts was divided
into two parts.
Phase 1 included the development of methods to determine the
presence of a) total coliforms (MPN), b) fecal coliforms (MPN),
c) Salmonellae, d) Shigellae, e) coagulase positive staphylo-
cocci, f) Protozoa (Entamoeba histolytica and Entamoeba coli),
g) Ova of Cestodes (Taenia sp., Diphyllibotrium latum, Diphy-
lidium caninum, Echinococcus sp.) h) Ova of Nematodes (Ascaris
lumbricoides, Strongyloides sp., Enterobius vermicularis,
Trichuris trichiura), i) Histoplasma capsulatum, j) Candida
albicans, k) Aspergillus fumigatus, and 1) Enteric viruses
(ECHO, Cocksackie, and Polio).
Phase 2 included the development of methods to determine the
survival patterns of and the percentage reduction of the
following organisms inserted in the windrows by the thermo-
physical processes or by antagonistic action caused by micro-
bial competition or by antibiotic inhibitors: a) bacteria:
Escherichia coli, Salmonella tvphimurium, Shigella sonnei,
Staphylococcus aureus (coagulase-positive), b) parasites;
Entamoeba histolytica, Ascaris lumbricoides (viable ova);
c) fungi: Histoplasma capsulatum Aspergillus fumigatus,
Geotrichum candidum; d) viruses: Poliovirus, e) spiro-
chaeta: Leptospira Philadelphia.
The aerobic biodegradation of organic solids and liquids to
a relatively stable end product comprised primarily of humus
is a satisfactory method for the disposal of solid waste with-
out creating health hazards and water, land or air pollution
(16). Since the finished product is an excellent soil condi-
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tioner, (17, 20, 26) aerobic thermophilic decomposition of
urban, refuse and sewage sludge may be applied for recycling
of municipal waste.
Debate over the use of compost as a soil conditL oner varies
from those who advocate its usefulness and safety (4, 5, 8,
13, 16, 17, 19, 20, 22, 23, 26) to those who question its
usefulness or safety (9, 21). However, it has been gen-
erally assumed that a correctly managed composting process
is adequate in destroying the diverse flora of pathogenic
microorganisms present in such an environment. Previous
studies have indicated that microorganisms are killed as a
result of the high temperatures obtained during composting.
However some investigators have indicated that an antibiotic
action is responsible for destruction of some pathogens (26).
Knoll (16) reported killing of Salmonella species within 2 to
7 days at compost temperatures of 122 F (50 C). At tem-
peratures below 112 F (45 C) destruction of all Salmonella
strains cannot be assured. In a later report Knoll (17)
found that typhoid bacteria were destroyed in compost at a
temperature of 112 F after 7 to 9 days ani in ampules up to
257 days and that these bacteria can resist the complex
antagonistic processes in composting material for 247 days.
Niese (19) concluded from his studies that the self-heating
of compost is dependent upon the availability of nutrients
and not upon the numbers of microorganisms present, but the
speed at which the maximum temperature is reached is in-
fluenced by the total number of microorganisms present.
Farkasdi (10) reported that rapid environmental changes in-
itially enhance the development of large numbers and vary-
ing species of microorganisms followed by a heating period
necessary for the destruction of pathogenic organisms. He
also indicated that the presence of fungi in compost is re-
lated to its moisture content. The drier the compost the more
numerous the fungi. The mesophilic fungi are numerous at
98 F (37 C), numerous thermophilic fungi at higher tempera-
tures and a complete absence of fungi at 150 F (67 C).
Rohde (22) found that Ascaris ova were destroyed after 4 days
in decomposing slaughter house wastes. He reported that
Ascaris ova are killed in 10 days at 104 F (40 C) and with-
in 24 hr at 140 F (60 C). Scott (23) reported that Endamoeba
histolytica, Endamoeba coli, and Ascaris ova are destroyed
within three weeks in windrow composting. Strauch (25 ) re-
ports that the psittacosis virus is inactivated when exposed
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to the environmental conditions in composting that are lethal
to pathogenic bacteria.
Studies by Sliepcevich (24) indicated that the handling and
disposal of raw solid wastes constitutes an improper health
hazard. She found from a study of the Department of Sanita-
tion of New York City that arthritis, cardiovascular diseases,
muscle and tendon diseases, and skin diseases could be
classified as occupational diseases of refuse collectors.
Anderson (1) also stated that the disposal of solid waste
is fundamentally a health problem.
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MATERIALS AND METHODS
The Johnson City open windrow composting plant processed city
refuse from which as much of the nonbiodegradable materials
as possible were removed (15). The refuse was ground into
small particles of from 1 to 3 in. in diameter, mixed with 3
to 5% primary dewatered sewage sludge and deposited by dump
truck in windrows approximately 4 to 5 ft high, 7 to 10 ft
wide and 150 ft long. The windrows were turned mechanically
at specified intervals of several days to insure proper aera-
tion and thorough mixing. The moisture content was maintained
at 50 to 60%. The composting process was usually completed
within 49 days.
A variety of specimens, including raw refuse, raw sewage, di-
gested sludge and sewage sludge-refuse from the windrow area,
were received from the Johnson City plant. U. S. Public
Health Service personnel were responsible for collecting all
specimens, including the methods for collecting samples, the
type of samples and the number of samples obtained from each
windrow. Figure 1 illustrates a typical cross section of the
windrow and the locations from which samples of compost were
obtained for examination.
Sampling procedures. A windrow was cut through from top to
bottom to expose two cross sections. Samples were taken from
each section of the windrow at the locations shown in Figure
1. Samples were collected with tongs which were sterilized
in the field between each use. Certain samples were combined
such as A with A-^; B with B-1-; etc. Samples were placed in
sterile plastic bags for transportation.
To insure uniformity and compatibility of all data, dry weights
of all specimens were determined and the results are expressed
as the number of microorganisms per g dry weight.
Dry weights were determined by placing a sample of known weight
in a vacuum oven at 176 F (80 C) for 2 to 5 days until all of
the moisture was removed as indicated by the consistent weight
of the sample. The percent moisture of each specimen was
calculated from the dry weights.
Due to the heterogeneous nature of compost, preliminary ex-
periments were carried out to determine the most accurate
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CUT THROUGH WINDROW
TYPICAL CROSS-SECTIONS
FIG. 1. Cross sections and sampling locations. A = 2-4 in.
below surface, B = 6-8 in. below surface, C = 2 ft below surface,
D = 6 in. above bottom, E = 2 in. above bottom.
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method for preparing a homogeneous aqueous suspension of the
specimens in order to get consistent results. Approximately
70 to 100 g random samples of compost were obtained in plastic
bags. After thoroughly mixing, 5 g sub-samples were sus-
pended in 95 ml of saline (0.85% salt solution), 0.1 M phos-
phate buffer at pH 7.0, and distilled water. Suspension of
the compost sample in each of these diluents were effected by:
1) vigorous manual shaking in a standard 100 ml dilution
bottle; 2) mechanical shaking on a rotary shaking machine
describing a circle one in. in diameter at 200 rpm for a
period of one min; 3) homogenizing in an Oster blender set
at slow speed (stir) for one min. Triplicate plate counts
were made on each suspension.
Of the three diluents tested, saline and 0.1 M phosphate buf-
fer gave consistently higher and more reproducible results
than did distilled water. It was decided therefore to use
saline as the standard diluent because of the large quantities
required, simplicity in preparation and less chance of error.
For suspending the compost in the diluent, homogenizing gave
consistent higher and more reproducible counts than did manual
or mechanical shaking and was therefore adopted as the stand-
ard means for suspending compost samples throughout the study.
Microbiological procedures
1. Total and fecal coliforms: Coliform counts were determined
by suspending 5 g weight samples of compost in 95 ml of sterile
saline (1-20 dilution). Duplicate 5 g samples were also
taken for dry weights determinations. Each suspension was
homogenized for 1 min, and processed according to procedures
described in Standard Methods for the Examination of Water
and Wastewater (2) by the use of the 5 tube technique for de-
tecting and quantitating total and fecal coliform densities.
All results are expressed as the number of organisms per g
dry weight of sample.
2. Fecal streptococci: The presence of fecal streptococci
was determined by inoculating aliquots of the serially di-
luted compost suspensions into duplicate tubes of KF Strepto-
coccal broth. This procedure was selected as being more con-
ducive than the KF Streptococcal agar when the two media
tested simultaneously gave compatible results. All tubes of
KF Streptococcal broth were checked for the presence of growth
after 48 hr incubation at 95 F (35 C). Periodic examinations
10
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of the growth in these tubes were made to confirm the pres-
ence of streptococci.
3. Coagulase positive staphylococci: In selecting a medium
for the isolation and identification of Staphylococcus from
compost, Chapman-Ston^ TPEY, Staph 110 and 5% blood agar
were compared. We found that colonies of Staphylococcus
isolated from compost were more easily recognized on Staph
110 than they were on the other types of media. Appropriate
dilutions (102, 104, and 106) of the compost suspensions were
plated on Staph 110 medium, incubated at 98.6 F (37 C) for
24 to 48 hr and the small, round, glistening, entire, yellow
to golden pigmented colonies were subcultured and subsequently
tested for coagulase activity.
4. Salmonella and Shigella: For the detection of Salmonella
and Shigella species direct plating proved to be unsatisfac-
tory, since a prohibitive amount of overgrowth was exhibited.
Dilutions of the compost suspension, in an attempt to over-
come excessive growth, resulted in uniformly negative results.
Therefore, selective enrichment media were used: Selenite
F enrichment broth and Selenite brillant green enrichment
broth containing sulfapyridine (SBG sulfa). Of these two
media, the SBG sulfa enrichment was more selective. Thirty
g of the compost samples (undiluted) were placedinto 270 ml
of enrichment media and incubated at 106 F (41.5 C) in a
controlled temperature water bath for 18 to 24 hr. After
incubation several loopsful of the enrichment broth culture
were streaked, in triplicate, on Salmonella-Shigella (SS)
agar, bismuth sulfite (BS) agar and MacConkey agar respec-
tively. All plates were incubated at 95 F (35 C) for 18 to
24 hr and checked for typical and suspected colonies of
Salmonella and Shigella. Suspected colonies were transferred
to triple sugar iron (TSI) agar slants, incubated at 95 F
for 24 hr and examined for typical reactions indicative of
Salmonella and/or Shigella species (7). Suspected positive
cultures from TSI agar were inoculated into urea broth,
lactose broth, and semi-solid agar. All cultures whose
biochemical reactions were typical of Salmonella or Shigella
species were submitted to the Tennessee Department of Public
Health Laboratories, Nashville, Tennessee for final sero-
logical identification by standard procedures recommended
by the Center for Disease Control.
11
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5. Total plate count: Five grains of compost were suspended
in 95 ml of sterile saline and homogenized at lowest speed for
1 min. From this initial suspension, dilutions of 10^ through
108 were inoculated into sterile petri plates in replicates
of eight. Since preliminary experiments had shown comparable
results using trypticase soy agar (TSA) and Standard methods
agar (SMA) as plating media, TSA was chosen as the media to
be routinely used. These plates were incubated at 95 F (35 C)
and at 131 F (55 C) both aerobically and anaerobically for
48 hr to determine the total number of colonies. Anaerobic
conditions were achieved via Gas Pak Anaerobic jars.
6. Enteroviruses: Approximately 20 to 30 g of each sample
received from the Johnson City Composting Plant were frozen
in sterile plastic bags, packed in dry ice, and shipped air
mail to Tennessee Department of Public Health Laboraotries in
Nashville. Upon receipt of the frozen specimens in the State
Laboratory, each sample was allowed to thaw completely at
room temperature, and the contents of the same bag were mixed
thoroughly by manipulation of the bag. Approximately 2 g
of the sample were removed and placed in a flask containing
20 ml of cold, sterile distilled water and glass beads. Pre-
liminary examinations indicated a 2 g sanpte was adequate.
The flask was shaken vigorously to mix the contents and the
suspension was poured into a sterile centrifuge tube. The
suspension was then clarified by centrifugation in a refrig-
erated centrifuge (4 C) at 1500 rpm for 20 min. The super-
nate was poured off and re-centrifuged for 1 hr at 3000 rpm.
The clear supernate was removed from the sediment and an
antibiotic solution was added to give a final concentration
per ml of 1000 units of penicillin and 1000 ug of strepto-
mycin. The sample was held at room temperature for 30 min.
The sample was then inoculated into 3 tubes of primary monkey
kidney cells (African Green purchased from Microbiological
Associates) and 3 tubes of Hep 2 cells (maintained by serial
passage in the State Laboratory). The tubes were incubated
on a roller drum at 98.6 F (37 C) for 8 to 9 days. All cell
cultures were observed daily for virus activity (6).
7. Parasites: Three methods were employed in the parasito-
logical examination of the compost samples: 1) direct mount;
2) brine flotation; and 3) formalin-ether sedimentation. To
prepare the samples for examination, 2 g of compost were
placed in a 250 ml flask containing 20 to 30 ml of saline aid
12
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enough glass beads to cover the bottom of the flask. After
thoroughly shaking to emulsify the sample, the flask was
tilted and left undisturbed for at least 30 min to allow
sedimentation.
Approximately 0.05 ml of the sediment was placed upon a
clean slide for direct microscopic examination (direct mount)
Iodine was employed to obtain characteristic differentation.
Following this procedure, the original suspension was
thoroughly mixed and strained through a wire mesh funnel
into a 50 ml centrifuge tube. The screen was washed with
saline and the final volume in the centrifuge tube was
brought to approximately 45 ml. After centrifugation at
2000 rpm for 1 to 2 min, the supernatant was decanted and
the sediment resuspended in 20 ml of saline. Ten ml of this
suspension was removed and saved for formalin-ether sedi-
mentation proceudre. The remaining 10 ml was centrifuged at
2000 rpm for 1 to 2 min and decanted. The sediment was re-
suspended in approximately 20 ml of brine (1.2 sp. gr.) and
transferred to a shell vial. The vial was filled to the lip
with brine and a clean glass slide was carefully superimposed
over the vial, taking care to avoid overflow and air pockets.
This preparation was allowed to stand 10 to 15 min and the
slide was carefully removed and fitted with a cover glass.
The edges were sealed with Vaspar and microscopic examina-
tion was made. This constituted the brine flotation method.
The remaining 10 ml of suspension was centrifuged at 2000 rpm
for 1 to 2 min, and the supernatant decanted. Approximately
10 ml of 10% formalin was added and after thorough mixing,
the suspension was allowed to stand for 5 min. Three ml of
ether was added and the tube was shaken vigorously for 30
sec and centrifuged at 1500 rpm for 1 to 2 min. Four layers
resulted from this centrifugation; a small amount of sediment
containing the ova and parasites, a layer of formalin, a
layer of debris, and a layer of ether. The top three layers
were carefully decanted and a sample from the bottom layer
was used to prepare an iodine mount for microscopic examina-
tion.
8. Pathogenic fungi: Five grams of compost samples were
added to 100 ml of sterile physiological saline. After
shaking to suspend the sample, the supernatant was separated
13
-------
and centrifuged at 2500 rpm for 15 min. The supernatant was
decanted and the sediment was thoroughly mixed and added to
sterile screw cap vials containing 10,000 units of penicillin
and 10 mg of streptomycin. This suspension was allowed to
stand at room temperature for 20 min.
Each of three white Swiss mice (4 to 6 weeks of age) were
inoculated intraperitoneally with 0.5 ml of the concentrated
sediment. At the end of 3 weeks, the mice were sacrificed
and a portion of the liver and the entire spleen were re-
moved and placed in a sterile petri dish. After these
tissues were minced, small portions were used to inoculate
two tubes of Sabouraud's agar and two tubes of Sabouraud's
agar containing 0.5 mg of Actidione (cycloheximide) per ml
and 0.05 mg of chloromycetin per liter. All cultures were
incubated for 4 weeks, with weekly examinations being made.
Smears of suspicious colonies were made and fungi were
identified by cultural characteristics.
Two tubes of Sabouraud's agar and two tubes of Sabouraud's
agar containing 0.5 mg of Actidione per ml and 0.05 g of
chloromycetin per liter was inoculated with a small portion
of the concentrated sediment. All tubes were incubated at
25 C, and examined weekly. At the end of 6 weeks, smears of
suspicious colonies were made and identified by cultural
characteristics (6).
Insertion studies
1. Bacteria: Insertion studies were carried out using cult-
ure tubes of Salmonella typhimurium, E. coli, coagulase posi-
tive Staphylococcus aureus, and Shigella sonnei to determine
their survival time in refuse-sludge compost windrows. To
study the effect of temperature, as well as the possible
effect of an antibiotic activity that might be involved in
the disappearance of pathogenic and indicator organisms from
compost, the test organisms were placed in closed containers
as well as in open contact with the compost.
Three methods were used to study the effect of temperature
on the inserted bacteria:
a. Young, 2-12 hr agar slant cultures in 10X75mm cotton
plugged test tubes were inserted at various depths
for varying periods of time in the windrows.
b. Young, 18-hr nutrient broth cultures in 16Xl25mm
screw cap tubes were also inserted in the windrows.
14
-------
c. Young, 18-hr nutrient broth cultures were sealed
in ampules and inserted into the windrows.
Three methods were used to study the possible effect of
antibiotics which might be present in compost.
a. One ml of 18-hr nutrient broth cultures were in-
jected into compost enclosed in small nylon bags.
b. One-to-two ml of 18-hr nutrient broth cultures
were placed on sterile cotton balls and inserted
directly into the compost.
c. Sterile filter paper discs were saturated with
young, 18-hr broth cultures of the organism
and placed directly into compost.
To facilitate the recovery of samples inserted into the
windrows, small nylon bags were used. Freshly prepared 0
day compost was placed in the bags along with the culture
tube (slants, screw cap tubes or ampules). The cotton balls
or filter paper discs were placed in the bags in direct
contact with compost. The bags were securely tied and in-
serted into the windrows. Sufficient quantities of these
"bagged" samples were prepared to allow four samples to be
taken from each depth (2-4 in., midpoint, or bottom) within
the sample period.
Recovery of organisms from broth and agar slant cultures:
Standard plate counts were run on all broth cultures re-
covered from the windrows. Agar slant cultures recovered
from the windrows were scraped with sterile inoculating
loops and streaked on nutrient agar plates.
Recovery of organisms from filter paper discs: 12. coli,
and Staphylococcus aureus were recovered from the compost
sample, placed in 100 ml of sterile saline, shaken vig-
orously by hand for one min and 0.1 ml amounts of the
saline suspensions were inoculated directly onto duplicate
plates of EMB and Staph 110 agar. Following 24 hr incuba-
tion, the plates were examined for typical colonies, which
were further confirmed by biochemical tests (7) .
Salmonella typhimurium: Filter paper discs recovered from
the windrows were placed in 100-200 ml SBG sulfa medium and
incubated at 106 F (41.5 C) for 18-24 hr. Duplicate plates
of SS agar and bismuth sulfite agar were streaked from the
15
-------
SBG sulfa and examined for typical colonies after 48 hr.
Those cultures which warranted further examinations were
inoculated into SIM agar, urea broth, and lactose broth.
If the results from these indicated the culture to be a
Salmonella the culture was sent to the Tennessee Department
of Public Health, Nashville, for serologicaltyping.
Shigella sonnei: The filter paper discs were recovered from
the windrow, placed in 100-200 ml of Selenite F broth, in-
cubation at 95 F (35 C) for 18-24 hr,and the procedures
used for the isolation and identification of Salmonella
typhimurium were carried out.
2. Enteroviruses. Suspensions of poliovirus (Type 2) were
received from the Tennessee Department of Public Health in
a frozen state. Portions of the liquid suspensions were
added to screw cap tubes as well as used to saturate filter
paper discs. The tubes and discs were placed in nylon bags
containing 0 day compost and inserted at all three depths
in the windrows. At various time periods, sample nylon
bags were removed from the windrows, frozen in dry ice and
mailed to the Tennessee Department of Public Health in
Nashville for viability studies.
3. Leptospira: Leptospira Philadelphia, obtained from
Women's Medical College in Philadelphia, Pa. was grown in
Fletcher medium base containing 10% leptospira enrichment
(Difco). Young cultures in screw cap tubes were inserted
into the windrows for various periods of time from 48 hrs
to 3 weeks. Upon removal of the culture tubes from the
windrows, viability of the organisms was determined by
making sub-cultures to fresh medium and observing the
growth under a darkfield microscope for motility. Filter
paper discs recovered from the windrows were placed in
16mm test tubes containing 3 ml of Fletcher medium base,
shaken manually for approximately one minute and the
supernatant examined under the darkfield microscope for
motile leptospira.
4. Fungi. Young sub-cultures of Histoplasma capsulatum,
Blastomyces dermatitidis, Geotrichum candidum, and
Aspergillus fumigatus were made on potato dextrose agar
or Sabouraud agar slants in 16xl25mm screw cap tubes and
inserted at various depths into the windrows. At various
16
-------
periods of time, these cultures were removed from the wind-
rows and sent directly to the Tennessee Department of Public
Health, Nashville, for positive identification and confirma-
tion of viability.
5. Parasites. Positive stool specimens were obtained from
area hospitals. One positive dog fecal specimen was also
included in the study. Each of the stool specimens were
divided into several equal samples in plastic bags and in-
serted in the windrow for varying periods of time. Upon
removal from the windrows, the samples were examined by the
Tennessee Department of Public Health, Johnson City branch
laboratory. Formalin-ether and salt flotation methods were
carried out to determine the presence (or absence) of ova
in the sample. Viability of the ova was not determined by
animal feeding.
Background studies
To better understand the bacterial ecology of composting a
study was carried out by two microbiology majors at East
Tennessee State University to examine the bacterial flora
of compost throughout the process and attempt to find an
organism more indicative of compost in the early stages
of decomposition than are the coliforms (11). These studies
were carried out by taking large samples of compost (50-
100 g) at 2 day intervals from 8-16 in. below the surface
at approximately midpoint in the windrow. A thoroughly
mixed 5 g sample was suspended in 45 ml saline, homogenized
for 1 min at slow speed, dilutions prepared in saline from
IQl through 109 and 0.1 ml of each dilution in duplicate
was spread evenly over the surface of SS, eosin-methylene-
blue, trypticase soy, and Staphylococcus 110 agar plates.
Duplicate plates were incubated at 95 F (35 C) and 131 F
(55 C) under aerobic and anaerobic conditions for 24 to
36 hr. The resulting colonies were observed macroscopically
and further identified by inoculating into appropriate bio-
chemical media, ie., TSI agar, SIM agar, citrate agar, urea
medium, and other differential media when required (7).
For comparative studies soil samples were obtained from
cultivated fields, pasture land and woodland areas. Five
g of each soil sample were examined for the presence of
total and fecal coliforms as well as for the presence of
Proteus. (12)
17
-------
RESULTS
The average number of total coliforms, fecal coliforms and
fecal streptococci found in raw refuse and sewage sludge
are shown in Figure 2. It is of importance to note that
the coliform count of raw refuse is equivalent to tha t of
sewage sludge and that the fecal streptococci count of raw
refuse is substantially greater than that of sewage sludge.
Season variations in the coliform counts of the windrows
was not observed. Although the results shown in Figure 3
would indicate that the coliform counts were somewhat less
during the Fall quarter of the year it should be pointed
out that during this period of time the project was just
getting underway, sampling procedures had not been estab-
lished, procedures for handling of the raw materials at the
composting plant were being investigated, and laboratory
procedures had not been standarized. Additional data that
could not be included in these average counts indicate that
the coliform counts remained fairly constant during the Fall,
Winter, Spring, and Summer seasons. Fecal streptococci
were made for the Fall and Winter seasons only.
Species of Salmonella were isolated occasionally from raw
refuse and sewage sludge only after 20 to 30 g of the
sample was cultured in an enrichment medium. Only on one
occasion was a Shigella isolated from sewage sludge. This
was early in the Fall of 1967 when the project was starting
and the species was not identified. The species of Sal-
monella isolated are listed in Table 1. In no instance
was Salmonella found in a windrow after 7 days.
The bacterial ecology of compost was found to be directly
related to the internal temperature of the windrow. The
freshly ground compost contains large numbers of micro-
organisms which utilize the carbohydrates present in re-
fuse as indicated by a characteristic drop in pH during
the first few days of the composting process. As a result
of the release of this excess energy the temperature of
the windrow reaches 120 F (49C) to a maximum of 167 F
(75 C) within 7 days and remains high throughout the pro-
cess. If this temperature is not maintained due to anaero-
biosis, the bacterial population, (especially the coliforms)
remains at a high level or increases in number. Figures 4,
-------
Refuse
Sewage Sludge
Refuse-sludge
FIG. 2. Average number of total coliforms, fecal coli-
forms and fecal streptococci in raw refuse, sewage sludge and
raw refuse-sewage sludge mixture on zero day. Numbers represented
averages of 6 to 8 specimens. Total coliforms ^| ; fecal
coliforms ^ ; fecal streptococci \\
19
-------
10y
IO8
10?
IO5
IO5
IO4
to
S io3
4-)
O
ro
cq 2
1CT
10-
Fall 1967
Winter 1968
Spring 1968 Summer 1968
FIG. 3. Average number of total coliforms, fecal coliforms and fecal
streptococci in refuse-sewage sludge on zero day for the four seasons. Data of
fecal streptococci for Spring and Summer not available. Total coliforms HI ;
fecal coliforms C^l ; fecal streptococci |~~1
-------
TABLE 1. Isolation of Salmonella from raw-refuse, raw-
sewage sludge and refuse-sewage sludge mixtures
on 0 day
Raw- Re fuse
Only
S. enteritidis
S. typhimurium
S. saint paul
S. heidelberg*
S. montevideo*
Raw- Re fuse Sludge
Only
S. enteritidis
S. typhimurium
S. saint paul
S. anatum**
S. Chester**
S. derby**
S. eimsfuettel**
S. muenchen**
Refuse- Sewage Sludge
Only
S. enteritidis
S. typhimurium
S. heidelberg
S. saint paul
S. braenderup***
*Isolated only from raw refuse, never found in raw sew-
age sludge.
**Isolated from raw sewage sludge, never found in raw
refuse.
***Isolated on one occasion from refuse-sewage sludge
mixture. It was not found in raw refuse or sewage sludge.
Salmonella were isolated from only a small percent of
the raw refuse or raw sewage sludge specimens examined.
21
-------
fa
0
4J
fO
SH
(D
80
66
52
38
24
10
fl
3
o
o
u
(0
-P
O
EH
(D
102L
10'
10
10-
10"
0
21
35
49
Time in Days
PIG. 4. Average total coliform MPN
from eight refuse-sewage sludge windrows. Vertical
bars represent extent of variations found in the
windrows. 2-4 in. depth.
22
-------
5, 6, 7, and 8 show the average total coliform counts pre-
sent throughout the 49 day refuse-sewage sludge compost
process. These counts are averages of the results obtained
from 8 windrows in an attempt to demonstrate the extent of
variations observed between" windrows and the various areas
within the windrow from which samples were obtained. These
results typify the extent of variation observed in samples
obtained 2-4 in. from surface (FIG. 4) and 2 in. from
bottom (PIG. 8).
The total microbial population in raw refuse and refuse
sludge windrow remain relatively constant throughout the
composting process as indicated by Figures 9 and 10. There
is however a complete change in the windrow flora. As the
temperature of the windrows increases the mesophilic bacte-
rial population is rapidly replaced with pseudothermophilic
and thermophilic bacteria, maintaining a remarkably constant
total bacterial count. The aerobic and anaerobic bacteria
population capable of growing at 95 F (35 C) increased in
number from lO^-lo^ on 0 day to approximately 10ฎ by the
49th day per g dry weight of compost.
The results of a more detailed study of the bacterial
ecology of compost are shown in Figures 11, 12, and 13. It
is evident from the results illustrated in Figure 11 that
although the coliforms disappeared from compost within
the first 2 weeks, they did reappear at various times
throughout the process period. This reappearance may be
due to recontamination with coliforms from the composting
plant equipment, flies and birds, or to the release of organ-
isms from organics during the decomposition process. The
fecal streptococci follow the same general pattern as the
coliform, but appear to be more resistant to the windrow
temperatures than did the coliform.
The mesophilic Bacillus species cannot survive the windrow
temperatures and disappear from the compost within two
weeks as shown in Figure 12. However, the pseudothermophi-
lic and thermophilic Bacillus species increase in number
after the first 2 weeks and remain throughout the process.
Numerous colonies of yellow-orange chromogenic bacteria
appear early in the process but disappeared rapidly as the
windrow temperature increased. These chromogenic bacteria
were short gram negative, gelatinase-producing bacilli be-
longing to the genus Serratia. During the second week
23
-------
175
fe 15ฐ
o;
3 125
tO'
s-i
0)
ง 100
EH
75
50
ง
-iH
C
ra
Cn
10
10
8
10
10"
10-
21
Time in Days
35
49
FIG. 5. Average total coliform MPN
from eight refuse-sewage sludge windrows. Vertical
bars represent extreme variations found in the
windrows. 6-8 in. depth.
24
-------
M
3
m
175
150
125
100
75
50
io94-
107J
lio6.
en
H
c
(D
? 10 5
o
ฐ104
10'
\
0
21
rtr
Time in Days
FIG. 6. Average total coliform MPN
from eight refuse-sewage sludge windrows. Vertical
bars represent extreme variations found in the
windrows. 2 ft depth.
25
-------
175
En 150
0)
3 125
ro
8, 100
e
0)
EH
75
50
-U
3= o-
10
to
co
H
c
ro
I
10'--
105
10
10-
10'
10-
4
0
21
35
49
Time in Days
FIG. 7. Average total coliform MPN
from eight refuse-sewage sludge windrows. Vertical
bars represent extreme variations found in the
windrows. 6-8 in. from bottom.
26
-------
175
150
125
cu
5 100
re
75
50
8
s-i
.OJ
10
io
ฃ
to 106
Cn
o
10
10-
4
10-
0
21
35
49
Time in Days
FIG. 8. Average total coliform ปMPN
from eight refuse-sewage sludge windrows. Vertical
bars represent extreme variations found in the
windrows. 2 in. from bottom.
27
-------
o
u
ro
H
n
0)
-p
u
ro
PQ
ro
-P
o
0
Time in Days
FIG. 9. Total plate count of raw refuse windrow 11-8-67 -
12-27-67- Each plate count was made from composites of 8 samples.
Plates prepared in duplicate and incubated at 95F and 13IF under
aerobic and anaerobic conditions. 95F aerobic , 1 ;
95F anaerobic 4 a, ; 131F aerobic a g ;
anaerobice> o
28
-------
10y
108
0
o
-U-
u
n)
ffl
2 10'
o
104
0
21
35
49
Time in Days
FIG. 10, Total plate counts of refuse-sewage sludge wind-
row. The refuse-sewage sludge windrow plate counts represents the
average counts obtained from three windrows. All plates were pre-
pared in duplicate and incubated at S5F or 13IF under aerobic and
anaerobic conditions. 95F aerobic ; 95F anaerobic^-A ;
131F aerobicj( _ _ ; 131F anaerobic o 0.
29
-------
10
200
TIME IN DAYS
FIG. 11. Survival time in a typical windrow (13-J) of total coliforms
fecal coliforms ; and fecal streptococci . Temperature g
-------
TIME IN DAYS
FIG. 12. Survival time in a typical windrow (13-J) of Bacillus sp.
Chromogenic sp. ; and pigmented Pseudomonads . Temperature *
-------
chromogenic colonies reappeared in significant numbers in
the compost and remained evident for the duration of the
process. These organisms were found to be gram positive
to gram variable, spore forming pseudothermophilic or
thermophilic Bacillus. Typical blue-green pigmented Pseudo-
monas colonies were found in the compost after 4 to 7 days
but disappeared in 18 to 24 days.
It was of interest to find that species of Proteus and
coagulase-positive staphylococi could be isolated in large
numbers from raw refuse but were never found in raw sewage
sludge. These organisms disappeared rapidly, however, as
the compost temperature increases (FIG. 13). Eighty per
cent of the Proteus strains isolated were found to be J?.
mirabilis, P. vulgaris was never isolated from any samples
examined.
The presence of Proteus in raw refuse and its disappearance
from compost, closely paralleling the disappearance of
pathogenic enteric bacilli inserted in the windrows, suggests
that this bacillus could be utilized as an indicator organ-
ism of raw refuse.
Total coliform an fecal coliforms were isolated from all
of the soil samples examined whereas Proteus was present
only in those soil samples containing decaying matter
(Table 2). When raw refuse was added to garden soil Proteus
could be isolated from the area only as long as the refuse
remained. When the refuse decomposed and disappeared from
the soil, Proteus could no longer be recovered.
Parasites have been observed, by direct end concentrated
wet mounts preparations, periodically from concentrated
sewage and refuse-sewage sludge compost. The parasite
ova most frequently observed were those of Ascaris, hook-
worm, Trichuris, Trichostrongylus sp. and H. diminuta.
Forty-two percent of raw dewatered sewage and first stage
sludge samples examined contained at least one parasite
protozoan ova or cyst. Parasites were observed in 33% of
the finished (49th day) compost samples. None of the sam-
ples were heavily infested, most contained only one parasite.
Of .the total number of samples examined, parsites were
observed in only 5-8% of the specimens. The parasites ob-
served in the finished product were characteristic of
those associated with bird and animal infestations and
32
-------
1011
4J 9
S 10
10
1 10
H
a t
<ง 10
ง 10
en
w 4
9 10
10
10
10
4
200
180
|160
40
Cu
0
120 |
100 |
&
80 ^
60
40
20
12
16
20
24
28
32
38
TIME IN DAYS
FIG. 13. Survival time in a typical windrow (13-J) of Proteus
coagulase-positive staphylococci . Temperature A
; and
-------
TABLE 2. Proteus and coliforms in soil
Soil Sample
Pasture
Garden
Woodland
Coliforms*
Total MPN Fecal MPN
6.6xl05
2.2xl05
1.6xl03
9.8xl03
6.6xl02
4-lxlO1
Proteus*
Negative
Negative
Positive
*Per 5 grams soil sample
34
-------
were not of human origin.
During the course of this investigation enteroviruses were
never isolated from sewage sludge, raw refuse or refuse-
sewage sludge mixtures. Subsequent investigation revealed
that no enteroviruses had been isolated by the Tennessee
Department of Public Health Department from human fecal
specimens submitted from the Upper East Tennessee area
from April 1964 to July 1, 1968. A large number of Echo
9 viruses were isolated in 1958 and 1959 (6). It is not
unexpected, therefore, that these viruses could not be
isolated from compost.
Pathogenic fungi could not be demonstrated in any of the
specimens of refuse-sewage sludge compost submitted to
the Tennessee Department of Public Health Laboratories in-
dicating either their absence in compost or their presence
only in extremely small numbers.
The results obtained from the insertion studies confirm
previous findings that the bacterial population of the wind-
row is directly related to its internal temperature. We
were unable to demonstrate any type of antagonistic action
resulting from antibiotic activity or other metabolic pro-
ducts of microorganisms in compost. Aqueous, ether, chloro-
form, alcohol, benzene or combined solvent extracts of
large quantities of compost, at various stages in the
process, were neither bactericidal nor bacteriostatic for
a wide variety of gram positive and gram negative bacteria.
The gram negative pathogenic enteric bacilli do not sur-
vive the normal windrow composting environment. Species
of Salmonella or Shigella originally present in refuse
or sewage or inserted under controlled conditions into the
refuse-sewage sludge windrows disappeared from the windrows
within 7 to 21 days. Figure 14 shows the survival of _E.
coli, jS. aureus, _S. typhimurium, and Sh. sonnei inserted
in a windrow in January, 1969 when the temperature of the
refuse-sewage sludge mixture was approximately 25 F (-4 C).
The temperature of the windrow did not reach 120 F (49 C)
until about 21 days. Under these conditions the cultures
not only survived but actually increased in number through
the 14th day. As the temperature approached 120 F the
bacterial population declined. While the Salmonella and
Shigella species were completely eliminated by the 28th
35
-------
E. coli
S. aureus
Sal, typhimurium
Shig. sonnei
ON
0
l\
\ป i
u
n
V '"
V
V
/ป /
/ \vX
x\\
\
\\
V,
u
\i
u
I
150
^
.IOC fl)
125 a
o
(D
i nn ?L
J.UU ft
C
K
(D
75 4
50
25
28
Time in Days
PIG. 14. Survival of microorganisms inserted in refuse-sewage sludge windrow. 1-7-69 to
2-4-69. Sealed culture vials inserted in windrow at 2 in. and mid-depth. Vials were removed
on days indicated and viable plate counts made of surviving bacteria. 2 in. depth
mid-depth ; 2 in. temperature . ; mid-depth temperature .
-------
day, E_. coli and _S. aureus cells were still viable at the
2 in depth on the 28th day. The temperature at the 2 in.
depth did not reach 120 F until approximately the 28th day.
Figure 15 illustrates the survival of these same micro-
organisms inserted in a windrow on a warm September day
when the temperature of the windrow was slightly over 100 F
(38 C) by the time the cultures were inserted. All the
microorganisms were completely eliminated by the 4th day
when the temperature rapidly reached 140 F (60 C). The
results of survival studies carried out with the four
bacteria in sealed tubes and on saturated filter paper
discs are shown in Table 3. Also shown are the results
obtained from the insertion of Geotrichum candidum and
Aspergillus fumigatus. It is obvious from these results
that despite the somewhat erratic temperature patterns of
the top 2 to 4 in. and the bottom 2 to 4 in. sections of
the windrow, all bacteria were killed by the 27th day,
and the fungi by the 35th day. In all of these studies
the microorganisms inserted in windrows which followed
a normal temperature pattern were rapidly killed. The
results in Table 4 are included to examplify the extreme
variation observed in one of the windrows. The tempera-
ture of this windrow was erratic and as a result E. coli,
JL typhimurium and Sh. sonnei survived through the 24th
day. Poliovirus was inactivated by the 7th day. One
culture of Geotrichum candidum survived 24 days and
Aspergillus fumigatus, inserted after the windrow was 14
days old, was killed within 10 days. These results are
of interest because this windrow was perhaps the most
erratic of all the windrows studied. However noe of the
organisms survived 27 days in spite of the irregularities.
Leptospira Philadelphia did not survive the windrow tempera-
ture for more than 2 days. Cultures of Blastomyces derma-
tidis and Histoplasma capsulatum generally did not survive
a 7 day exposure to the windrow as illustrated in Table 5.
Cysts of Endolimax nana and Entamoeba histolytica, as
well as hookworm ova (Necator americanus or Ancylostoma
duodenale) obtained as stool specimens from hospital
patients disappeared (disintergrated) after 7 days exposure
to the windrow environment (Table 6). On the other hand,
as shown in Table 7, hookworm, tapeworm and whipworm ova
from dog feces were observed in specimens 35 days after
37
-------
10
S. aureus
E. coli
S. typhimuriijm
Shig. sonnei
175
150
125 CD
(D
100 2
ct
C
(D
75 *
50
125
J 4 3 b U 1 /i J 4 b b
Time in Days
FIG. 15. Survival of microorganisms inserted in refuse-sewage sludge windrow
9-5-68 to 9-12-68. Sealed culture vials inserted in windrow at 2 in. and mid-depth.
Vials were removed on days indicated and viable plate counts made on surviving bacteria.
2 in. depth ; mid-depth 2 in. temperature
mid-depth temperature.
-------
TABLE 3. Survival of microorganisms inserted in refuse-sewage sludge windrow
1-23-69 to 2-27-69
Day
0
7
14
27
35
Depth
2"
Mid
Bot.
2"
Mid
Bot.
2"
Mid
Bot.
2"
Mid
Bot.
Temp.
F.
102
122
136
88
110
96
144
152
96
122
142
98
E. coli
Tube
4.2xl07
^ioi
^lO1
^lO1
-
-------
TABLE 4. Survival of microorganisms inserted in refuse-sewage sludge windrow
1-17-68 to 2-13-69
Day
0
7
14
24
27
Depth
2"
Mid
Bot.
2"
Mid
Bot.
2"
Mid
Bot.
2"
Mid
Bot.
F
110
120
140
124
148
140
70
118
64
146
148
102
E. coli
7.3xl07
l.SxlO8
1.6xl05
^lO1
^lO1
^lO1
<10i
3.5x104
"- 101
7.5xl02
^ 101
^ 101
^ 101
s.
aureus
2.9xl07
4.8xl07
l.SxlO5
8.5xl02
^ ^lO1
^lO1
x-10-L
z_ 101
/. 101
^.10^
z. 101
s- 10 x
^- 101
s.
typhimurium
4.2xl07
S.OxlO6
^-lO1
^lO1
-ilO1
+ 101
^ 101
S.OxlO2
+ 101
-i 101
X
<<- lof
-/101
Sh.
sonnei
3.5xl07
3.1xl03
+ 101
1.6xl02
-c 101
-i. 101
-clO1
l.OxlO2
*~ 101
S.OxlO2
^1 10-1
/- 101
^lO1
Polio
Virus
Type II
3000
TCID50
-
G.
candidum
+
X
+
+
X
X
X
+
X
A.
fumigatus
Inserted
after
windrow
was
14 days
old
+
+
+
-
= No growth; + = growth; X = contaminated or broken tube. Discs saturated with cultures
of the bacteria listed in the Table were negative by the 7th day.
-------
TABLE 5. Survival of microorganisms inserted in refuse-
sewage sludge windrow 10-21-68 to 11-12-68
Day
0
2
3
4
7
9
14
22
Depth
2"
Mid
2"
Mid
2"
Mid
2"
Mid
2"
Mid
2"
Mid
2"
Mid
Temp . F
110
96
138
124
114
120
124
144
140
154
142
158
128
152
L.
Philadelphia
+
-
X
X
-
_
B.
dermatidis
+
Specimens
taken only
from 7th
to 22nd
days
-
_
H.
capsulatum
+
Specimens
taken only
from 7th
to 22nd
days
-
-
_
+ = growth; - = no growth; X = broken or lost in windrow
41
-------
TABLE 6. Survival of human parasites inserted in refuse-
sewage sludge windrows 9-5-68 to 10-2-68
Day
0
7
14
21
28
Depth
Midpoint
Midpoint
Midpoint
Midpoint
Temp . F
140
153
159
141
Cysts
E. nana
+++
-
-
-
-
E. histo-
lytica
++
-
-
-
-
Hookworm
ova
+++
-
-
-
-
+++ = Heavy infestation-many cysts or ova observed in wet mount;
++ = several cysts or ova observed in wet mount; - = no cysts
or ova observed in wet mount.
42
-------
TABLE 7. Survival of dog parasites inserted in
refuse-sewage sludge windrows 12-11-68 - 1-20-69
Day
0
6
20
27
35
Depth
2"
Mid
2"
Mid
2"
Mid
2"
Mid
Temp . F
137.5
159
120
150
124
Ova
Hookworm
+++
+++
+++
X
+++
+++
+++
+++
X
Tapeworm
+++
+++
+++
+++
+++
+++
+++
+++
X
Trichurius
+++
+++
X
+++
+++
+++
X
+++
X
+++ = Heavy infestation; large numbers of intact ova observed
in wet mounts.
43
-------
being inserted into windrows. While these ova were present
in large numbers it was not possible to determine their
viability. It would appear from these results that parasi-
tic ova and cysts from human infections are more suscep-
tible to disintegration by the composting process than are
dog parasitic ova. Although parasitic ova may remain in-
tact they may no longer be viable. Unfortunately funds
were not available to complete this phase of the study.
44
-------
REFERENCES
1. Anderson, R. J. 1964. The public health aspects of
solid waste disposal. Public Health Reports. 79:93.
2. Anon. 1971. Standard Methods for the Examination of
Water and Wastewater, 13th ed. American Public
Health Association, New York.
3. Anon. 1972. Mission 5000-a citizens' solid waste
management project. An Environmental Protection
Publication in the solid waste management series
SW-lists.
4. Amrami, A. 1958. Agricultural utilization of sewage
and public health problems. Tavruah (Association for
Promotion of Sanitation in Israel). 1(2 and 3):
26-40, April.
5. Banse, H. J., G. Farkasdi, K. H. Knoll and D. Strauch.
1968. Composting of Urban Refuse. International
Research Group on Refuse Disposal Information Bul-
letin, No. 32.
6. Barrick, J. H. 1968. Tennessee Department of Public
Health, personal communication.
7- Breed, R. S., E. G. D. Murray, and N. R. Smith. 1957-
Bergey's Manual of Determinative Bacteriology. 7th
ed. Williams & Wilkins Co., Baltimore
8. Davies, A. G. 1960. The composting of refuse. The
Sanitarian (Br.) 68 (1):19-22, Oct.
9. Editorial. 1960. Compost's value overrated. Public
Health (Johannesburg) 15:70.
10. Farkasdi, G. 1961. Part I: Biological processes in
composting urban refuse. 1. Contribution on the
microbiology of composting. International Research
Group on Refuse Disposal Information Bulletin. Nti.
13:2.
45
-------
11. Gaby, N. S., L. C. Creek, and W. L. Gaby. 1970.
Utilization of Proteus as an Indicator Organism in
Composting. J. Enviro. Health, 32:559.
12. Gaby, N. S., L. C. Creek, and W. L. Gaby. 1971.
A study of the bacterial ecology of composting and
the use of Proteus as an indicator organism of solid
waste. Developments in Industrial Microbiology.
Proc. of the 28th general meeting-Fort Collins, Colo.
13. Hanks, T. G. 1967. Solid Waste/Disease Relationships:
A Literature Survey. U. S. Public Health Service
Publications No. 999-U14-6.
14. Jansen, J. and Kunst, H. 1958. Are pathogenic micro-
organisms killed in waste dumps where sufficiently
high fermentation temperatures occur? Netherland J\
Agr. Sci. 1:111-114.
15. Kochtitzky, 0. W., W. R. Seaman, and J. S. Wiley.
1969. Municipal Compost Research at Johnson City,
Tennessee. Compost Sci., j^:5.
16. Knoll, K. H. 1959. Composting from the hygenic
viewpoint. International Research Group on Refuse
Disposal Information Bulletin. No. 7:142.
17. Knoll, K. H. 1963. Influence of various composting
processes on non-sporeforming pathogenic bacteria.
International Research Group on Refuse Disposal In-
formation Bulletin. No. 19:1.
18. Morgan, M. T. and F. W. Macdonald. 1969. Test show
M B Tuberculosis doesn't survive composting. J.
Envir. Health. 32:101.
19. Niese, G. 1963. Experiments to determine the degree
of decomposition of refuse compost by its self-
heating capability. International Research Group
on Refuse Disposal Information Bulletin. No. 17:1.
46
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20. Parrakova, E. 1962. Hygenic criteria for the evalua-
tion of refuse compost. International Research Group
on Refuse Disposal Information Bulletin, No. 16:10.
21. Reeves, J. B. 1960. Sanitary aspects of compost
sewage sludge and sawdust. Sew, and Ind. Wastes
31 (5):577-64, May, 1959. Abstract 364 in Supple-
ment 2: Composting of Organic WastesAn Annotated
Bibliography, J. S. Wiley, USPHS Publication, p. 64.
22. Rohde, et.al. 1957. Summary of disucssions at
meeting of AKA in Dusseldorf: Destruction of patho-
gens during composting. International Research
Group on Refuse Disposal Information Bulletin, No.
3:61.
23. Scott, J. C. 1953. Health aspects of composting
with night soil. WHO, Expt. Comm. on Enviro. Sanit.
3rd session, Geneva.
24. Sliepcevich, E. M. 1955. Effect of work conditions
upon the health of the uninformed sanitationmen of
New York City. Doctoral dissertation. Springfield
College, Springfield, Mass.
25. Strauch, D. 1964. Requirements of veterinary hygiene
in the removal of urban refuse. International Re-
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26. Wiley, J. S. 1962. Pathogen survival in composting
municipal wastes. Journal Water Pollution Control
Federation. 34:80.
47
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-670/2-75-023
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
EVALUATION OF HEALTH HAZARDS ASSOCIATED WITH
SOLID WASTE/SEWAGE SLUDGE MIXTURES
5. REPORT DATE
April 1975; Issuing Date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO,
William L. Gaby
9. PERFORMING ORG '\NIZATION NAME AND ADDRESS
Department of Health Sciences
East Tennessee State University
Johnson City, Tennessee
10. PROGRAM ELEMENT NO.
1DB064;ROAP 24ALU;Task 03
11. CONTRACT/GRANT NO.
68-03-0128
12. SPONSORING AGENCY NAME AND ADDRESS
National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
Contact - Clarence A. demons 513/684-4481
16. ABSTRACT
This report summarizes and evaluates the health hazards associated with
municipal solid waste/sewage sludge composting by the windrow composting
process. The occurrence and survival of pathogens, parasites, and indi-
cator bacteria at various stages during the composting process are
described. The study shows that windrow temperatures of 120F to 167F
(49C-74C) maintained for at least 7 days destroy pathogens and human
parasites. Dog parasitic ova, however, remain intact 35 days after
exposure. Considerable variation in the temperature is found at the top
and bottom 2 inches of the windrow indicating that proper turning of the
compost is essential to ensure destruction of pathogens and parasites.
It is concluded that a properly composted solid waste or solid waste/
sewage sludge mixture is microbiologically acceptable as a soil condi-
tioner for gardens, farms, and lawns, or for filling areas of erosion
without creating health hazards.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Ib.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Composting
Survival
Parasites
Sewage
Sludge
Solid waste
Sewage sludge
Pathogens
Health aspects
13B
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
56
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
48
.S. GOVERNMENT PRINTING OFFICE:, 1975-657-592/5359 Region No. ,5-1
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