SAND 78-1304
Unlimited Release
Moisture Effects on Inactivation and
Growth of Bacteria and Fungi in Sludges
(Presented at the National Conference on Design of Municipal
Sludge Compost Facilities, Chicago, August 29-31, 1978)
Jerome R. Brandon, K. Sieglind Neuhauser
repared by Sanaa Laboratories, Albuquerque,
and Livermore, California 94550 for the United States Department
of Energy under Contract ATI29-11-789
Printed September 1978
>F 29000(7-73)
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SAND78-1304
MOISTURE EFFECTS ON INACTIVATION AND GROWTH OF BACTERIA AND
FUNGI IN SLUDGES
J. R. Brandon
K. S. Neuhauser
Applied Biology and Isotope
Utilization Division 4535
Sandia Laboratories
Albuquerque, NM 87185
ABSTRACT
Inactivation of bacteria and fungi by either heat or ionizing radiation
is dependent on moisture content of the sludge system. In general,
bacteria and fungal spores exhibit more resistance to inactivation when
treated in the dry state. Moisture levels below 20 percent are not
supportive of bacterial growth in composted sludge. High levels of
coliforms effectively inhibit growth of salmonellas and of Aspergillus
fumigatus in composted sludge systems.
This work was supported by the Division of Advanced Systems and
Material Production Division, U. S. Department of Energy, Washington,
DC, and the Municipal Environmental Research Laboratory, U. S.
Environmental Protection Agency, Cincinnati, OH, Interagency Agreement
E(29-2)-3536/EPA-IAG-D6-0675.
3-4
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TABLE OF CONTENTS
Introduction
Page
7
Materials and Methods
7
Results and Discussion
8
Radiation Inactivation
8
Heat Inactivation
10
Drying-Bed Simulation Experiments
13
Growth in Composted Sludge
17
Inhibition of Growth in Composted Sludge
20
Summary
23
References
24
5-6
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The sewage sludge irradiation program at Sandia Laboratories has
included treatment of liquid and solid sludges.
Considerable data on
liquid sludges, raw as well as digested, have been generated.
For
studies of the value of sludge as a cattle feed supplement, however,
we have irradiated dried raw and dried digested sludges.
In addition,
a significant portion of our recent efforts has dealth with pathogen
reduction in composted sludge.
Our primary research goal is the determination of parameters for
inactivation of pathogens by ionizing radiation and/or heat.
The
effects of reduced moisture levels on these rate parameters may be
important.
This paper presents very recent data in this area for
bacteria and fungi as well as results of experiments on growth (and
the inhibition of growth) of bacteria and fungi in composted sludge,
a low-moisture system.
MATERIALS AND METHODS
Determinations of bacterial populations were made by standard
plating techniques described elsewhere.l,2 Fungal counting involved
preparation of the appropriate dilutions and plating on potato-dextrose-
yeast extract agar containing chloramphenicol.
For the radiation inactivation studies, a cobalt-60 gamma source
was used, at dose-rates ranging from 10 to 120 kilorads absorbed energy
per minute.
Heat inactivation experiments were carried out in water
baths at appropriate temperatures.
In most cases one-gram samples of
sludge or saline containing bacteria were sealed in 1" x 1" plastic
packets to allow for rapid heat transfer.
Upon removal from the water
bath, the packets were placed in ice.
Proper dilutions of liquid
samples were then prepared and plated.
Solid or semi-solid samples
were mixed with 9 ml of saline and blended at high speed for 30 seconds
and appropriate dilutions were then plated.
In some cases, especially
7
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for fungal studies, the samples were contained in l3-mm test tubes
during the experiment, and reconstituted with saline using a Vortex
mixer.
Drying experiments were performed in aluminum trays, outdoors in
the sunlight.
Relative humidity averaged 10-15 percent.
Ambient tem-
peratures ranged from a low of l5-20°C at night to a high of 32-38°C
during the day.
RESULTS AND DISCUSSION
Radiation Inactivation
It had been suspected for some time that the sensitivity of bac-
teria to ionizing radiation may depend to some degree on their state
of desiccation.
Table 1 shows results of a recent experiment wherein
the "starting" materials were samples of sludge which had been air-
dried to ~90 percent solids but which still contained relatively high
populations of viable bacteria.
(In the case of salmonellas, the
sludge had been autoclaved and inoculated with a mixed salmonella cul-
turej the salmonellas were then allowed to multiply in the liquid
sludge to high levels prior to drying) .
3 d. 4
In contrast to their behavior in liquid systems an In compost,
the bacteria in the dried samples exhibited a wide range of sensitivi-
ties, probably due to their varying states of desiccation within indivi-
dual samples (heterogeneity within these dried samples was unavoidable).
The most striking case is seen to be that of coli forms, where the
apparent D-value is ten-fold higher (arrows)
for two samples than that
measured routinely in liquid systems.
This resistance to inactivation
is seen to a lesser extent in the case of fecal streptocci (three fold
difference in one case) and salmonellas (generally somewhat higher).
An attempt was made to determine whether desiccation effects were
in fact responsible for the sporadic increase in resistance in these
dried samples.
Radiation inactivation curves for saline suspensions
8
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Table 1
Irradiation of Dried Raw and Digested Sludges
Apparent Apparent
Species D-valuea D-Valuea
and Dose in Dried in Dried
a Raw Sludqe Digested Sludge
D-Values b (krads)
in Liquids (krads) (krads)
20 17 18
40 19 18
Coli forms 60 16 16
(20-30 krads) 80 30 30
100 322 -- -
120 32 -
240 353 -- -
60 67 50
100 67 45
Fecal Strep 150 75 52
(120-130 krads) 240 185 92
360 360 -- -
480 100 -
40 67 31
60 35 -
Salmonellas 80 38 38
(25-30 krads) 100 48 45
150 58 50
240 77 -
a.
D-value is the dose required to effect a 90 percent
reduction in population.
The indicated range of D-values applies to both raw
and digested sludges.
b.
9
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of mixed coliforms, one intact and one having been desiccated by stream-
ing dry air over it are shown in Figure 1.
Clearly there is a signi-
ficant difference in the "wet" and "dry" sensitivities.
Even though
glycerol was present in both samples, and may have provided some degree
of radiation protection, results of a subsequent experiment without
glycerol indicated that this increased resistance is due to the desicca-
tion effect.
Several species of the fungus Aspergillus are commonly found in
5
sewage sludge or sludge products.
Aspergillus flavus is of concern
because of its potential for aflatoxin production.
Aflatoxins are
potent carcinogens.
This is of particular interest in our cattle feed-
ing program.
Aspergillus fumigatus is generally thought of as a
"secondary" pathogen, usually affecting individuals with a primary
infection or who are immunologically compromised.
The latter fungus
is found wherever aerobic decomposition occurs and its potential for
causing harm may have been somewhat overplayed recently.
Nevertheless,
it is of interest to know how these species respond to the treatment
conditions inherent in the Sandia Laboratories program, namely, ionizing
radiation.
Our studies have shown that spores of A. flavus in dried
sludge are inactivated at a rate of approximately 50-60 kilorads per
log (data not shown), a O-value in the same range as those for coliforms
or salmonellas.
Our radiation inactivation results agree fairly well
. 6
with those previously reported by others in liquid suspenslons.
It is
expected that A. fumi~atus will respond similarly; this determination
as well as that of the effects of increasing moisture on this O-value
are currently being made.
Heat Inactivation
It had been shown earlier that the sensitivities of some enteric
7
bacteria to heat differed in compost and liquid sludges.
Figure 2
shows this difference for salmonellas at 50°C.
Although some differ-
ence might be expected between a mixed culture and a single strain, it
10
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should not account for the 5-log difference (105 reduction) observed.
The effect of 61°C heat was determined for sludge samples that had
been air-dried but which still had relatively high residual populations
of coliforms and fecal streptococci.
This was done in order to compare
the sensitivities of these bacteria to those of the normal flora in
liquid sludges, as well as to those of similar bacteria allowed to grow
to high levels in composted sludge.
Figure 3 shows that in the dried-
sludge system coliform bacteria are much more resistant to heat than
are coliforms allowed to grow in composted sludge.
Limited data for
liquid sludge show that the indigenous coliforms are even more sensitive
here than in compost.
A qualitatively similar effect is seen for fecal
streptococci (Figure 4), even though these bacteria are known to be
generally hardier than coliforms or salmonellas.l Experiments are
planned to determine whether a sludge component(s) which would be con-
centrated during drying provides this protection or whether the resis-
tance indeed depends simply on degree of desiccation.
The effects of heating Aspe~gillus flavus in dried sludge and in
aqueous suspension are shown in Figure 5.
Again, protection seems to
be provided by the dried system.
More than an hour at 60°C is required
to reduce the population by 99.9 percent, while in the aqueous suspen-
sion, 99.99 percent viability is lost in 5-10 minutes (a four-log reduc-
tion is approaching the limit of detectability in this system).
Drying-Bed Simulation Experiments
Even though dried sludges appear to protect bacteria or fungi from
the effects of heat or ionizing radiation, few data are available on
the effects of the desiccation process itself in inactivating these
microorganisms.
In order to predict how effective air-drying in the
arid Southwest is in reducing pathogen levels in sludges, experiments
were set up to monitor the populations of several species as a function
of time and of moisture content.
13
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30 60 90 120
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TIME ,MINUTES
Fig',~---" 3.
Heat inactivation 161°C) of coliforms in
liquid digested, 8 percent solids (0); in
composted, -60 percent solids I~); and in
dried digested, >90 percent solids Ie)
sludges.
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20
30 40
50
60
TIME, MINUTES
Figure 5.
Heat inactivation (61°C) of Aspergillus
flavus conidia in aqueous suspension (0)
and in dried raw sludge (8).
16
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The systems used were as follows:
(A)
Coliforms and fecal streptococci in raw and in digested
sludges;
(B)
Salmonellas allowed to multiply to high levels in
sterilized raw and digested sludges;
(C)
Raw sterile and raw untreated sludges inoculated to
high levels with Aspepgillus flavus.
In all cases, the sludge (initially 8-10 percent solids) was placed
5 em deep in trays, and was mixed thoroughly at each sampling time
(about twice per day), in order to minimize heterogeneity within the
samples.
In actual drying beds at a treatment plant, mixing is not
done.
For coliforms, fecal streptococci, and A. flavus the outdoor dry-
ing was not very effective in reducing the populations.
The data for
Aspepgillus are presented in Figure 6; this curve represents the maxi-
mum population reduction of the three species, and at the end of the
3
experiment there were still >10 spores/g.
In a subsequent experiment
(data not shown), viable coliforms were still found after three weeks
and viable fecal streptococci were found after five weeks; fungi were
not monitored.
On the other hand, the drying process appears to significantly
reduce the salmonella populations, as shown in Figure 7.
This is not
unexpected because these bacteria are known to be more fastidious than
coliforms or fecal streptococci.
It is interesting that the "break"
in the curve occurs at about the time the sludge reaches 80 percent
solids.
Growth in Composted Sludge
A potential problem associated with sludge treatment and disposal
is the possibility of repopulation by pathogenic bacteria or fungi fol-
. 1 d d8,4
lowing a disinfection process. It had been prevlous y emonstrate
17
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Figure 6.
18
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TIM E t DAYS
4
Effect of sludge drying on conidia of
A. flavus aoded to normal raw sludge (0)
and sterilized raw sludge (8). Initial
population was -5xl05 conidia/gram.
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DAYS
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TIME t
Figure 7.
Effect of sludge drying on mixed salmonella
allowed to grow in sterilized raw (.) and
in sterilized digested (0) sludges. The
sludges reached 80 percent solids midway
through day three. Initial populations were
-109 bacteria/gram.
19
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that Salmonella enteritidis ser. Montevideo bacteria can grow rapidly
in composted sludge, in spite of the fact that the moisture content is
only approximately 40 percent.
It was therefore important to determine
the minimum moisture level that will permit such growth.
Sterilized
compost samples were dried to various degrees, inoculated with mixed
salmonellas, to a level of -4xI03/g and incubated for 24 hours at 37°C.
The data presented in Figure 8 indicate that rapid growth to high levels
can occur at moisture levels greater than approximately 20 percent.
Thus, a 20 percent moisture content seems to represent a critical
threshold; significant repopulation after disinfection should not occur
in composted sludges at or below this moisture level.
Inhibition of Growth in Composted Sludge
It was recently demonstrated that saturating composted sludge with
mixed coliforms effectively inhibits growth of salmonella bacteria.9
An experiment was conducted to determine if these "innocuous" bacteria
could likewise inhibit growth of Aspergillus sp. in compost.
Comparing
curve A with B in Figure 9 indicates that A. fumigatus is only par-
tially inhibited.
However, curve C shows that the normal microbial
flora of the composted sludge prevent its growth.
It is of interest to
note the effect of 45°C incubation on the growth (Curve B').
The ini-
tial growth rate is greater than that of the A. fumigatus incubated at
20°C.
This is expected, since A. fumigatus normally prefers somewhat
higher temperatures for growth.
However, after one week the fungal
population drops, even though this temperature is much lower than those
normally reached in a compost operation (frequently >65°C).
This could
be the result of either depletion of a critical nutrient(s) or the
accumulation of a toxic metabolite.
20
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108
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Figure 8.
---
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,
NORMAL
COMPOST\
I I
40
o
10
I
20 30
MOISTURE CONTENT
Effect of moisture content of composted
sludge on growth potential of salmonella
bacteria. Samples were inoculated at
-4xl03 bacteria/g and incubated at 37°C
for 24 hours.
21
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22
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SUMMARY
Moisture levels of the sludge systems studied can greatly influence
the survival, growth, and resistance to heat and radiation inactivation
of bacteria and fungi found in these systems.
Some enteric bacteria
seem to behave almost like spore-formers, i.e., microorganisms which,
under duress, go into a dormant, highly-resistant state.
It must be stated that many of these results are preliminary.
Studies are currently underway to further elucidate the mechanisms by
which protection from heat and from ionizing radiation is afforded in
dried sludges.
Growth kinetics work is also continuing.
It is hoped
that the results reported herein will stimulate similar work on the
effects of moisture on growth and inactivation of bacteria and fungi
in sludge systems.
ACKNOWLEDGMENT
The authors sincerely thank Sara L. Langley and Jean Womelsduff
of the Applied Biology and Isotope Utilization Division for their
extraordinary efforts in obtaining the data reported herein.
23
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24
REFERENCES
1.
Brandon, J. R., and S. L. Langley, 1976.
"Inactivation of Bacteria
in Sewage Sludge by Ionizing Radiation, Heat, and Thermoradiation,"
SAND75-0168, Sandia Laboratories, Albuquerque, NM.
2.
"Standard Methods for the Examination of Water and Wastewater,"
13th Edition, 1971.
DC, p. 691.
American Public Health Association, Washington,
3.
"Waste Resources Utilization Program Interim Report, 1976.
SAND76-0350, Sandia Laboratories, Albuquerque, NM.
4.
(a) Brandon, J. R., W. D. Burge, and N. K. Enkiri, 1977. "Inactiva-
tion by Ionizing Radiation of Salmonella entepitidis ser. Montevideo
Grown in Composted Sewage Sludge," Appl. Environ. Microbiol. 33,
pp 1011-1012. (b) Brandon, J. R., and S. L. Langley, unpublished
data.
5.
Cooke, W. B., and P. Kabler, 1955. "Isolation of Potentially
Pathogenic Fungi from Polluted Water and Sewage," Public Health
Rep. Wash. 70, pp. 689-694; and Cooke, W. B., 1957. "Check List
of Fungi Isolated from Polluted Water and Sewage," Sydowia !'
pp. 146-175.
6.
Mohyuddin, M., and N. Osman, 1974. "Modification of the Radiation
Resistance of Aspepgillus flavus Conidia by Some Chemicals,"
Radiation Botany li, pp. 23-27-
7.
"Effect of Heat on Patho-
Ward, R. L., and J. R. Brandon, 1977.
genic Organisms Found in Wastewater Sludge," Proc. of the 1977
National Conf. on Composting of Municipal Residues and Sludges,
Information Transfer, Inc., Rockville, MD., pp. 122-127.
8.
Burge, W. D., and N. K. Enkiri, USDA/SEA, Beltsville, MD., unpub-
lished results.
9.
Brandon, J. R., and S. L. Langley, manuscript in preparation.
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