United States
Environmental Proteciton
Agency
Water Engineering
Research Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-86/106 Mar. 1987
&EPA Project Summary
Regrowth of Salmonellae in
Composted Sewage Sludge
W. D. Burge, P. D. Millner,
N. K. Enkiri, and D. Hussong
Research was conducted to investi-
gate the regrowth of salmonellae in
composted sewage sludge. Though
composting effectively stabilizes and
disinfects sewage sludges, the de-
crease in salmonellae may be only tem-
porary, because this pathogen can sur-
vive and grow without a human or
other animal host.
Modification of an agar medium im-
proved our ability to detect salmonellae
in composts. Salmonellae were de-
tected in four composts from 30 com-
posting sites across the United States.
However, all composts supported
salmonella growth when sterilized by
radiation. These results and those by
others suggest that the microflora in
composts suppress salmonella growth.
To determine the nature of salmo-
nella suppression in composts, we in-
vestigated the effects of groups of the
compost microflora and the character-
istics of the substrates used by salmo-
nellae in composts. Results indicated
that suppression of salmonella re-
growth is mainly a result of bacterial
competition for a limited number of
substrates that these organisms use in
common with salmonellae.
This Project Summary was devel-
oped by EPA's Water Engineering Re-
search Laboratory, Cincinnati, OH, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Composting is a very effective proc-
ess for stabilizing and disinfecting
sewage sludge. The high temperatures
achieved in the composting process in-
activate pathogenic organisms and re-
sult in population densities that ap-
proach or are below analytical detection
limits. For viruses, certain bacteria, and
parasites requiring specific hosts for
survival, inactivation results in a perma-
nent decrease in their densities. For
salmonellae, which can propagate in
the absence of specific hosts, the reduc-
tion in numbers may be only temporary.
Repopulation of compost by salmonel-
lae may occur through regrowth of the
organisms existing in the compost at an
undetectable concentration or through
the growth of organisms introduced
from an outside source. A likely source
may be feces from salmonella-infected
birds, reptiles, or other animals.
Salmonellae infecting these animals are
also infectious to humans. Thus even
though the composting process
achieves treatment conditions that
meet the further pathogen reduction
criteria set forth in 40 CFR Part 257,
"Criteria for Classification of Solid
Wastes Disposal Facilities and Prac-
tices: Interim, Final, and Proposed Reg-
ulations" (as corrected in the Federal
Register of September 21, 1979); there
may still be a potential for repopulation
of composted sewage sludge by salmo-
nellae.
Anecdotal and a few scientific reports
of salmonellae in composted sewage
sludge have been made. Studies using a
few composts have indicated that sal-
monellae can grow extensively only if
the compost has been sterilized. This
finding indicates that the microflora
present in composts prevent salmonella
regrowth through antagonistic effects
that are not understood.
To evaluate the potential for salmo-
nellae to grow in sewage-sludge com-
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post, we modified an agar medium used
in the most-probable-number (MPN)
method to improve our ability to detect
salmonellae and used it to assay the sal-
monella content and salmonella growth
potential of sewage-sludge composts
collected from 30 compost sites across
the United States.
The factors involved in preventing
growth were studied by methods de-
signed to segregate the microbial popu-
lations of the compost on the basis of
temperature growth range and other
physiological and biochemical proper-
ties, so that individual and groups of or-
ganisms could be tested for their antag-
onistic capabilities.
To gain information on the number of
soluble, usable substrates involved in
salmonella regrowth, kinetic studies of
salmonella growth in composts were
conducted and analyzed according to
Monod's growth equations.
Modified Agar Method for
Detecting Environmental
Salmonellae by the MPN
Method
Methods of detecting and enumerat-
ing low numbers of salmonellae from
environmental samples have used MPN
methods, which require careful selec-
tion of colonies from a plated agar
medium. Xylose lysine brilliant green
(XLBG) agar was modified to control the
loss of selectivity caused by heating the
brilliant green component. The agar
content was increased to reduce colony
spreading. Brilliant green (BG) dye and
reagents to form the H2S indicator were
added after cooling the medium to 50°C
and just before pouring. H2S-positive
salmonellae were easily distinguished
from most other gram-negative bacteria
present in sewage sludge compost.
Salmonella recovery from compost
increased strikingly as a result of the
suppression of competing organisms
when BG dye was added after autoclav-
ing. In previous analyses of composts
and sewage sludges using brilliant
green (BG) and bismuth sulfite (BS)
agars, only 7% of the salmonella-like
colonies picked were confirmed bio-
chemically and serologically as salmo-
nellae. In analyses using commercial
XLBG agar, 27% of the colonies picked
were confirmed as salmonellae. How-
ever, salmonellae were detected using
BG and BS agars in two of the samples
that had been negative using XLBG
agar. In recent surveys of 15 composts
using the XLBG agar in which the BG
dye was added after autoclaving, 21 of
26 (81%) of salmonella-like colonies
picked were confirmed biochemically
and serologically as salmonellae.
The use of modified XLBG agar has
resulted in fewer nonsalmonellae being
picked for further MPN analysis and has
greatly reduced the work load associ-
ated with the MPN method. Direct plat-
ing was possible for enumerating
salmonellae in laboratory composts
containing about 103 or more salmonel-
lae.
Growth of Salmonellae in 30
Composted Sewage Sludges
Sewage sludge composts from 30
municipalities were sampled, and 4
samples (12%) contained salmonellae.
Salmonellae inoculated into the com-
posts died out unless the compost had
been sterilized. In radiation-sterilized
composts, the salmonellae grew.
Growth and death rates were found to
be moisture and flora associated. The
growth and death rates for antibiotic-
resistant salmonellae were the same as
those of nonresistant strains. In non-
sterile air-dry composts, salmonellae
persisted longer than in nonsterile
moist composts. It was concluded that
the active, indigenous flora of composts
establish a barrier to colonization by
salmonellae, and that in the absence of
competing flora, reinoculated salmonel-
lae may grow to potentially hazardous
densities.
Microbial Suppression of
Salmonella Regrowth
Recent studies of a few composts and
the studies of this report have indicated
that the microflora of composts sup-
press the regrowth of salmonellae. In
this work, compost microflora were ex-
amined for the antagonistic effect of in-
dividual microorganisms and groups of
microorganisms on salmonella growth
in compost.
Compost microflora from different
temperature zones in compost piles
were compared for their abilities to in-
hibit salmonella growth. Pure culture
isolates of compost microbes were
tested individually in agar plates and in
groups in sterile and experimental com-
posts to determine their contribution to
suppression. The microflora were re-
moved from the compost in extracts,
fractionated by centrifugation and filtra-
tion, and reintroduced into sterile com-
post to compare the activities of the dif-
ferent fractions on salmonella growth.
Of several hundred isolates from
compost, 23 bacteria, 61 actinomycetes^
and 42 fungi were chosen to represent a
range of morphologically and taxonom-
ically different compost microorgan-
isms. None of the bacteria or actino-
mycetes inhibited salmonella growth in
agar-plate inhibition assays. In contrast,
six fungal isolates did, but no growth
inhibition was evident when three of the
fungi, chosen because they expressed
the greatest antagonism, were inocu-
lated with or before salmonella into
sterile compost.
The capability of microorganisms
from different compost temperature
regimes to inhibit salmonella growth
was determined. Compost from the
70°C zone of a compost pile did not sup-
press salmonella growth. Compost
from a 55°C adiabatic incubator was
more suppressive, and compost from a
curing pile from a surface area that was
near ambient temperature was com-
pletely suppressive.
Studies involving the size fractiona-
tion of the flora obtained in compost ex-
tracts again showed the lack of the abil-
ity of fungi to suppress salmonella
growth and indicated that although the
actinomycetes suppressed growth to
some extent, gram-negative bacteria*
played a larger role. Of the gram{
negative bacteria, the coliforms were
much more effective than the noncoli-
form organisms.
Given the diversity of the microbial
population of cured compost at ambient
temperature, it was concluded that
salmonella regrowth would be negligi-
ble. Because total inhibition is not re-
lated to the activities of any single
group of microorganisms, no microbial
assay can be recommended to deter-
mine the capability of a compost to sup-
press salmonella regrowth.
Influence of Substrate on
Salmonella Regrowth
The kinetics of salmonella growth in
suspensions and extracts of irradiation-
sterilized composts were studied to de-
termine the number of substrates and
the relative amounts of the substrates
used. Three composts from widely sep-
arated compost sites in the United
States were used. Initial studies showed
that growth of salmonellae in suspen-
sions of compost did not appear to be
first order; but growth in extracts was
(p < 0.01), indicating a soluble substrate
and an insoluble substrate that became
solubilized as growth proceeded in the.
presence of the compost solids. m
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The magnitudes of the growth-rate
constants obtained using the extracts
were sensitive to the quantity of com-
post used up to a maximum amount,
and a hyperbolic relationship was found
when growth-rate constants were plot-
ted against the amount of compost ex-
tracted (Figure 1A). Plotting growth-rate
constants against the maximum
amount of salmonella growth brought
some of the outlying data points in
closer to the hypothetical curve (Figure
1B).
The linear forms of the hyperbolic
curves generated by the data all ap-
peared to fall on a single curve (Fig-
ure 2). The correlation coefficients for
the three curves all exceeded 0.997.
Models involving multiple versus single
parameters were tested for relative fit of
the data to the regression line of Fig-
ure 2. The correlation coefficient of a
model with three separate intercepts
and three separate regression coeffi-
cients was 0.9988, whereas that for one
combined equation (one intercept and
one regression coefficient) was 0.9977.
Although the multiple parameters im-
proved the fit, the difference was so
small that on the basis of parsimony,
the simpler model combining the three
equations to yield a single intercept and
a single regression coefficient was pre-
ferred.
Figure 1. Growth-rate constants (k, h V for salmonellae as plotted against: A. amount of
compost extract added and B. total amount of salmonellae gro win (compost 6175,
open circles; compost 6266, closed circles, and compost 6252, triangles).
2.0
1.6
1.2
0.8
0.4
—I ' ' —I ' ' f
0 0.4 0.8 1.2 1.6 2.0 2.4 2.8
Wscfu/mL
5.6
2.0
1.6
1.2
0.8
0.4
0 20 40 60 80 100 120 140
Compost mg/mL
200
The results of this study showed that
it is possible to extract a water-soluble
substrate from compost that will sup-
port first-order growth of salmonellae.
The first-order nature of the kinetics and
the combined data for the three com-
posts used suggest that there is a single
substrate among the composts support-
ing salmonella growth. The identifica-
tion of this substrate and the testing for
its presence in other composts might
possibly furnish valuable information
as to the factors involved in the re-
growth of salmonellae in composts.
Conclusions
Selecting salmonella colonies when
enumerating low numbers of salmonel-
lae in sewage-sludge and compost sam-
ples can be difficult because of the
growth of organisms that mimic salmo-
nellae. This difficulty can be greatly alle-
viated by modification of the standard
XLBG agar medium. The modification
involves using high concentrations of
BG dye that has not been heated be-
yond 50°C.
Studies of composts collected from
30 composting sites throughout the
United States show that inhibition of the
growth of salmonellae by the indige-
nous microflora of composts is a gen-
eral phenomenon.
When the complete microflora of
compost (bacteria, actinomycetes,
fungi, and protozoa) are present or in-
troduced into sterile compost, they fully
suppress the regrowth of salmonellae.
A major proportion of suppression
comes from the coliforms, with comple-
menting activity from other gram-
negative bacteria. Thermophilic and
mesophilic actinomycetes also supple-
ment the suppressive activity, but the
effect of fungi is negligible. The contri-
bution, if any, by protozoa was not de-
fined.
Three composts from widely sepa-
rated composting sites in the United
States contained water-extractable sub-
strates that supported the growth of
Salmonella typhimurium. Kinetic stud-
ies of salmonella growth indicate that
these substrates in the different com-
posts are very similar, if not identical,
and that total salmonella growth is a
sensitive assay for their concentration
in composts.
Recommendations
Modification of the xylose lysine (XL)
agar base (agar increased to 2% and 6 to
7 ppm BG dye added to the autoclaved
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Figure 2. A plot of the population and rate-constant data of Figure IB according to the
linear form of Monod's equation (see Fig. 1 for meaning of symbols).
7.5
1.0
0.5
medium after cooling to 50°C) appears
to provide a useful alternative to other
plating media for salmonella assay of
sewage sludges and sewage-sludge
composts. Increasing the BG dye con-
tent of the modified XLBG to 9 ppm was
found to increase the effectiveness in
discriminating for salmonella colony
growth. We suggest, however, that
each user run a study to determine what
concentration works best for salmonella
measurement. Additional studies are
recommended to compare the recovery
of salmonellae from similar samples
(i.e., sludges, composts) with other pro-
cedures. Also, comparison of the modi-
fied medium and other media to recover
indigenous salmonellae is recom-
mended.
The resident microflora in the com-
posts apparently provide a safety factor
preventing the colonization of sewage-
sludge composts by salmonellae. It has
been suggested that composts be steril-
ized by irradiation. We suggest that
complete sterilization may result in
unchecked growth of salmonellae if the
composts become inoculated. The pos-
sibility that partial sterilization may de-
stroy pathogens and yet inhibit salmo-
nella growth needs evaluation.
The fungi play essentially no role in
suppressing the growth of salmonellae
introduced into composts. Schemes to
prevent or control fungal growth can be
used if they do not eliminate gram-
negative bacteria, particularly coli-
forms, from the compost.
The findings that bacteria most
closely related to salmonellae play the
major role in suppressing salmonella
growth and that similar water-soluble
substrates in the three composts stud-
ied support salmonella growth suggest
that a study to determine the identity of
these substrates may furnish the key to
understanding and perhaps controlling
the regrowth of salmonellae in com-
posts.
Studies are recommended to deter-
mine the contribution of protozoans
and other parasites for suppressing sal-
monella regrowth in composted sew-
age sludge.
The full report was submitted in fulfill-
ment of Interagency Agreements EPA
No. DW21930587-01-3-1 and USDA
No. AD-12-F-4-A029 by the U.S. Depart-
ment of Agriculture under the partial
sponsorship of the U.S. Environmental
Protection Agency.
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W. D. Burge. P. D. Mi liner, and N. K. Enkiri are with the U.S. Department
of Agriculture, Beltsville, MD 20705; and D. Hussong is with the Maryland
Environmental Service, Annapolis. MD 21401.
Gerald Stern was the EPA Project Officer (see below for present contact).
The complete report, entitled "Regrowth of Salmonellae in Composted Sewage
Sludge," (Order No. PB 87-129 532/AS; Cost: $13.95, subject to change)
will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
For further information Albert D. Venosa, can be contacted at:
Water engineering Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45260
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EPA/600/S2-86/106
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