United States
Environmental Protection
Agency
Water Engineering Research
Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-86/047  June 1986
Project  Summary
Inactivation  of  Enteric
Pathogens   During  Aerobic
Digestion  of  Wastewater
Sludge

Samuel R. Farrah, Gabriel Bitton, and Stephen G. Zam
  A study was conducted to provide data
on the ability of aerobic digestion to re-
duce pathogens and to determine the ef-
fect of important variables. Laboratory and
field studies investigated the effects  of
aerobic and anaerobic digestion on enteric
viruses,  enteric bacteria,  total aerobic
bacteria, and intestinal parasites.  Under
laboratory conditions, the temperature of
the sludge digestion was the major factor
influencing survival of bacteria and vi-
ruses. The survival of both bacteria and
viruses was increased substantially by
decreasing the temperature of sludge di-
gestion from 28° to 7°C.  Lowering the
temperature or dissolved  oxygen level
reduced the percentage of solids-asso-
ciated organisms for bacteria but not for
viruses.
  Bacteria were inactivated at different
rates  during aerobic  sludge digestion.
Streptococcus faecalis was more stable
than Salmonella typhimurium or Escheri-
chia coll. Varying detention time or source
of sludge did not affect the rate of inacti-
vation of viruses or bacteria.
  Aeration of stock Ascaris suum ova in
0.1 N H2SO4 resulted in 91% embryon-
ation. However, aeration in sludge resulted
in only 19% to 50% embryonation after
30 days. Most of the ova embryonated in
0.1 N H2SO4 (79% to 93%) were infec-
tive for rats. In contrast, only 9% to 12%
of the ova embryonated  in  aerobically
digesting sludge were infective for rats.
  Full-scale aerobic digestion of sludge re-
duced densities of bacteria and entero-
viruses. In most  cases, the reductions
were  close to the maxima predicted for
completely mixed digesters that contin-
ually receive undigested sludge.
  The number  of parasitic ova varied
greatly with the community served by the
treatment plant. Parasitic  ova were re-
covered from most of the samples of di-
gested and undigested sludge from certain
treatment plants but not from others. The
relatively low numbers of ova recovered
from the sludge samples make it difficult
to evaluate the effects of sludge digestion
on these agents. The inactivation rate of
laboratory-grown bacteria  in aerobically
digested sludge was higher than that for
indigenous bacteria. The indigenous bac-
teria were probably better protected from
predation by protozoans and other an-
imals. Predation was the major factor in-
fluencing survival of both indigenous and
laboratory-grown bacteria during aerobic
digestion of sludge under laboratory con-
ditions. Predation had little effect on sur-
vival of viruses, however.
  This Project Summary was developed
by  EPA's Water Engineering  Research
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 order-
ing information  at back).


Introduction
  A large number of enteric bacteria, viral
pathogens, and  parasitic ova may be ex-
creted by infected individuals and may
therefore be present in untreated sewage.
Since a large number of these pathogens
become associated with wastewater so-

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 lids, many are not completely inactivated
 during sewage treatment processes and
 are merely transferred to wastewater slud-
 ges. These sludges are processed further,
 generally by aerobic or anaerobic diges-
 tion,  to  improve  their  dewaterability,
 reduce their unpleasant odor, and reduce
 their  pathogen content.
   The ability of  digestion processes to
 reduce  sludge pathogens has received
 considerable investigation, though num-
 erous data gaps exist. The information is
 particularly sparse for aerobic digestion.
 This investigation was undertaken to pro-
 vide  more definitive  information on the
 ability of  aerobic  digestion to  reduce
 pathogens and to determine the effects of
 important  variables.  The investigation
 studied the effects of aerobic digestion on
 pathogenic bacteria,  bacterial  indicator
 organisms,  parasitic   ova,  and  animal
 viruses.

 Experimental Procedures
   In laboratory experiments (which em-
 ployed temperature-controlled, laboratory,
 aerobic and anaerobic  digesters), cultured
 bacteria and viruses were generally used.
 In field studies,  conducted at existing
 plants in the vicinity of Gainesville, Florida,
 indigenous microbial species were used.

 Laboratory Experiments
   Aerobic digestion was conducted  in
 temperature-controlled vessels holding  15
 L  of  sludge. Sludge  was mechanically
 agitated, and humidified  air  was intro-
 duced through spargers. Anaerobic diges-
 tion was conducted in externally heated
 carboys holding 8  L of sludge. Bacteria and
 viruses  charged  to the  digesters were
 grown from type  cultures. Ascaris suum
 ova were obtained from the feces  of
 naturally infected hogs. Detailed proced-
 ures for growing organisms and measur-
 ing densities are presented in the report.
   Survival of bacteria and viruses and the
 association  of these agents with sludge
 floes  were studied in  11 individual trials.
 During each trial, two to four digesters
 were operated under different conditions
 of temperature, dissolved oxygen, and de-
tention time. Aerobically digested sludge
 was obtained from one of three local treat-
 ment plants and aerated for 2 to 4 days at
the desired temperature before the exper-
iments were started, thus stabilizing the
sludge at the desired conditions before an
experimental trial was started. An anaero-
bic sludge digester was  started using
anaerobically digested sludge obtained
from Tallahassee. At the beginning of each
trial, bacteria and viruses were added  to
 each of the digesters to obtain an initial
 concentration of approximately 105/mL
 On each  subsequent day, a portion of
 sludge  was removed from each digester
 for analysis, and a portion of wasted
 sludge from the plant that was the source
 of  the  aerobically digested sludge was
 seeded with bacteria and viruses and add-
 ed  to the digesters. The wasted sludge
 that was used for the daily additions to the
 digesters  was obtained at the beginning
 of each trial and was kept at 4 °C without
 aeration. The volume  of digested sludge
 removed and replaced with wasted sludge
 was determined by the detention time de-
 sired: 1/15 to 1/40 the volume for 15- or
 40-day detention times, respectively.
  Laboratory studies were also conducted
 on the survival of seeded Ascaris suum in
 aerobically digested sludge. Three aeration
 procedures were used that produced dif-
 ferent degrees of mechanical abrasion on
 the ova. Aeration was provided by shak-
 ing 200 ml of sludge in 500-mL flasks on
 a reciprocal shaker, by mixing 200 ml of
 sludge in 1000-mL beakers using a mag-
 netic  stirrer,  or by  aerating  200-mL
 samples in  500-mL flasks with air  dif-
 fusers connected to a small air pump. All
 three  methods provided approximately 5
 mg/L  of dissolved oxygen.  The aerobic
 digestion was run under two conditions:
 batch operation and daily feeding to sim-
 ulate  operation  with  40-day residence.
 Samples of the sludge were examined for
 total  recovered ova, ova embryonation,
 and ova infectivity in  rats.

 Field Studies
  Sludge  samples from nearby waste-
 water treatment plants were obtained over
two periods: From January to December
 1981,  and  from August to October 1983.
The sludge samples were examined for the
presence  of enteric  bacteria,  enteric
viruses, and parasitic ova. The dissolved
oxygen, temperature, pH, and total solids
of the sludge samples were also deter-
mined. Unlike the laboratory experiments,
no organisms were seeded into the slud-
ges; only densities of  indigenous  micro-
organisms were measured.

 Results and Discussion

Bacteriological Investigations

 Laboratory Studies
  The influence of temperature and dis-
solved  oxygen  level on inactivation of
bacteria during  aerobic and anaerobic
sludge digestion was studied in  11 trials,
each lasting approximately 9 days. The re-
sults  from  one trial appear in  Table  1,
 which  presents the log daily change in
 bacterial densities. The log daily change
 is the difference between the logs of the
 bacterial densities of the sludge in  the
 digester shortly after introduction of the
 feed sludge containing the inoculum and
 the sludge withdrawn from the digester 24
 hr later (before addition of the next batch
 of  feed). Table  1  shows the effects of
 temperature  (28.3°  and  6.2°C)  and
 dissolved oxygen  concentration on the
 bacteria in one of the trials. These results
 are typical of those obtained in other trials.
 Wasted, aerobically digested sludge from
 a nearby plant  was added daily to the
 digesters to provide a detention time of 15
 days. Results  show much greater reduc-
 tions for S. typhimurium, S.  faecalis, and
 E. coli  than for P. aeruginosa and total
 aerobic bacteria. For all bacterial types, the
 log daily change was much lower at 6.2 °C
 than at  28.3°C (compare Conditions I and
 II in Table 1). The presence or absence of
 air  (compare Conditions I and III in Table
 1) produced a small effect that was usually
 not significant.
  The bacteria  in  the digesting sludge
 were either in the supernatant or were oc-
 cluded to solids. Operation at 28.3°C in
 the aerobic state showed a markedly dif-
 ferent distribution  of bacteria from  the
 other conditions, averaging about 8% in
 the supernatant  compared with 36% for
 the other conditions. Subsequent experi-
 ments confirmed that grazing by protozoa
 accounted for the lower proportion of bac-
 teria in  the supernatant  at  28.3°C and
 contributed to the greater daily reduction
 in bacteria obtained at these conditions.
  Subsequent trials  with sludges from
 three different  wastewater treatment
 plants sludges showed that the source of
 sludge  did not  significantly affect  the
 results.
  In all  of the previously described ex-
 periments, bacteria were added to the di-
 gesters  daily along  with fresh sludge. The
 values for change in total bacteria there-
 fore reflect changes that occurred in the
 first 24  hr following bacterial addition. To
 determine whether the rate of daily change
 in bacteria would  fall off with  time,  in-
 dicating the presence of a resistant frac-
 tion  of  added bacteria,  digesters were
 operated under aerobic conditions without
the addition of bacteria  or sludge.  Two
 digesters were operated at 28 °C  and one
 at 6°C.  All digesters were operated with
greater than 4 mg/L dissolved oxygen. Re-
sults with S. typhimurium, S. faecalis, and
total aerobic count  showed that curves of
log density versus time were approximate
ly linear (with  negative slopes) over  the

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Table  1.    Bacterial Survival and Association with Sludge Floes During Aerobic and Anaerobic Digestion of Sludge:  The Influence of Sludge
           Digestion Conditions on Individual Bacteria
                               Sludge                    Dissolved                      Total      Daily Change    Bacteria in
                              Digestion     Temperature     Oxygen                     Solids        in Total     Supernatant
       Bacteria                Condition*        (°C)          (mg/L)          pH           (g/L)         Log10          (%)
Salmonella typhimurium


Streptococcus faecalis


Escherichia coli


Pseudomonas aeruginosa


Total aerobic bacteria


1
II
III
1
II
III
1
II
III
1
II
III
1
II
III
28.3
6.2
28.0
28.3
6.2
28.0
28.3
6.2
28.0
28.3
6.2
28.0
28.3
6.2
28.0
2.7
3.7
0
2.7
3.7
0
2.7
3.7
0
2.7
3.7
0
2.7
3.7
0
6.0
7.4
6.3
6.0
7.4
6.3
6.0
7.4
6.3
6.0
7.4
6.3
6.0
7.4
6.3
20.6
19.6
18.4
20.6
19.6
18.4
20.6
19.6
18.4
20.6
19.6
18.4
20.6
19.6
18.4
- 1.24Ai
-0.238
-0.90A
-0.92A
-0.108
-0.83A
- 1.13*
-0.23°
-0.618
-0.28*
-0.29*
-0.34A
-u.34A
-0.078
-0.098
8C
23B
51 A
12B
50*
47 A
4B
25*
30*
3s
34*
47*
7B
21*
33**
* Condition I = aerobic; Condition II = aerobic; and Condition III - anaerobic.
^Figures in a single column with identical letter superscripts are not significantly different.
course of several days, indicating no per-
sistent fraction of bacteria. The rate of in-
activation was substantially lower in these
experiments than when inocula were add-
ed daily (compare Tables 1 and 2). The
greater reduction in supernatant bacteria
at the higher temperature (Table 2)  is con-
sistent  with the inoculum  experiments.

Field  Studies
  Summarized data from field studies are
presented in Table 3. Plants 1  and  2 used
aerobic digesters, and Plant 3 used an an-
aerobic digester. For the aerobic digesters,
greater bacterial reductions were achieved
in Trial 2 than in Trial  1. The probable ex-
planation for this difference was digestion
temperature: Trial 1 was conducted during
colder months. All of the digesters were
completely mixed. With complete mixing,
some sludge inevitably short-circuits to
the exit with relatively short-term treat-
ment. Bacterial reduction was calculated
assuming complete mixing and destruc-
tion of bacteria except for bacteria in that
portion of the feed that leaks through to
the exit. Log reductions calculated  on this
basis (labeled "maximum  possible") are
presented in Table 3  where they  can be
                                   compared with experimentally determined
                                   reductions. For Trial 2, experimental and
                                   calculated values are similar;  this result
                                   supports the assumptions of the calcula-
                                   tion and indicates that use of complete
                                   mix reactors may contribute to failure to
                                   achieve high bacteria or virus  reductions
                                   in digesters.

                                   Virus Investigations

                                   Laboratory Studies
                                     Laboratory studies determined the influ-
                                   ence of several variables on the survival
                                   of viruses during aerobic and anaerobic
                                   digestion of sludge under laboratory con-
                                   ditions. The variables studied included
                                   temperature, dissolved oxygen level,  de-
                                   tention time, virus type, and the source of
                                   the sludge used for digestion studies.
                                     The  sludge source did not significantly
                                   affect the rate of inactivation of poliovirus
                                   when  sludge  was  aerobically digested
                                   (15-day detention time). At 28 °C and dis-
                                   solved oxygen levels of 5 mg/L, the mean
                                   daily  change  (Iog10) in inactivation of
                                   seeded poliovirus (Type I, Lsc) for sludges
                                   from three sources ranged from -0.71 to
                                   -0.97,  which  was  not  a  significant
                                   difference.
Table 2
    Bacterial Survival and Association with Sludge Floes During Aerobic Digestion of
    Sludge without Addition of Fresh Sludge
                       Daily Change in Total         Bacteria in Supernatant
                          Bacteria Log10                      (%)

Bacteria
                               6°C
                                              28°C
Salmonella typhimurium        -0.15
Streptococcus faecalis          —0.05
Total aerobic bacteria          -0.07
                                     -0.48
                                     -0.23
                                     -0.19
 * Figures with the same letter superscript are not significantly different.
6°C
S38*
80A
41C
28°C
3°
2D
3°
  The temperature of sludge digestion in-
fluenced the rate of inactivation  of po-
liovirus  (Table 4), which was highest  at
28 °C, intermediate at 17.6°C, and  lowest
at 5.5 °C.
  Varying the dissolved oxygen between
1 and  6  mg/L at a relatively constant
temperature of 28 °C did not change the
inactivation rate.  The mean daily change
(Iog10) ranged from -0.77 to -1.03.  Anaer-
obic digestion of the same sludge at 32 °C
produced a mean daily change (Iog10)  of
-0.33, significantly lower than for aerobic
digestion.
  Varying the detention time of aerobic
digestion between 16 and 40 days did not
change  the inactivation rate  (log daily
change) of poliovirus.
  Poliovirus 1, echovirus 1, coxsackievirus
B3, and the simian rotavirus SA-11 were
all inactivated at similar rates during aero-
bic digestion of sludge at 28 °C. The range
of log daily change  was -0.46 to  -0.77.
  Aerobically digested  liquid sludge when
allowed to dry  in a centrifuge tube from
about 1 to 50 g/L over 28 days showed
essentially no  surviving viruses  (polio-
virus), whereas 5%  survived in a  tightly
capped  control.

Field Studies
  Viruses were detected in  samples  of
mixed  liquor solids and  aerobically  di-
gested  sludge. The lowest  levels were
found in sludge from the second aerobic
digester at the plants  studied. However,
the numbers of viruses were so variable
that statistical analyses did not show that
the differences were significant except in
a few cases. Results ranged from 0 to 20

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 Table 3.
 Treatment
  Plant*
           Summary of Field Data on Bacterial Reduction During
           Aerobic and Anaerobic Digestion of Sludge
                            Bacteria
                                                            Bacterial Reduction^
                                                                  (Log10)
                                                          Trial 1
                                                                        Trial 2
 Plant 1
 (aerobic digester)
Plant 2
(aerobic digester)
Plant 3
(anaerobic digester)
Total coliforms
Fecal coliforms
Fecal streptococci
Aerobic bacteria
Maximum possible1

Total coliforms
Fecal coliforms
Fecal streptococci
Aerobic bacteria
Maximum possible'

Total coliforms
Fecal streptococci
Maximum possible'
    Plant 1:  Influent sludge and second of two 8.5-day residence time aerobic
            digesters in series
    Plant 2:  Sludges in first and second of two aerobic digesters, each with
            50 days of residence time
    Plant 3:  Influent sludge and sludge from an anaerobic digester with  10
            days of residence time.
    Log bacterial density in digested sludge - log density in undigested sludge.
    January through December 1981.
    August through October 1983.
- 7.53
- 7.52
- 7.03
-0.35
- 1.86
- 1.02
- 1.09
-0.77
-0.80
- 1.70
	
—
—
- 1.99
—
- 1.63
—
- 1.86
- 1.92
—
- 7.77
_
- 7.70
-0.91
- 1.10
- 1.00
    Calculated assuming total destruction of bacteria except for leakage caused by complete
    mixing: 1:72.2 for Plant 1,  1:50 for Plant 2, and 1:10 for Plant 3.
Table 4.    Effect of Temperature on Poliovirus (Type 1, Lsc) Survival
           in Laboratory-Scale Aerobic Digesters
Temperature
28
17.6
5.5
Dissolved
Oxygen
(mg/L)
5.8
5.2
5.8
Total
Solids
(g/U
10.4
7.9
7.9
Volatile
Solids
(g/U
4.8
5.7
5.2
pH
5.3
6
5.3
Mean Daily
Change in
Virus Survival*
(Log10)
- 0. 77A
-0.5B
-0.21C
  Means within the same column with the same letter are not significantly different
  at the p = 0.05 level.
plaque-forming units (PFU/g) of sludge
solids.
  Poliovirus and coxsackievirus serotypes
were isolated from the different sludge
samples. Compared  with wasted sludge
before digestion, the aerobically digested
sludge contained relatively few types of
viruses.

Parasite Investigations

Laboratory Studies
  Three methods of mixing (magnetic stir-
ring, shaking, and aeration with air pumps)
were used to test the effects of aerobic
sludge digestion on the embryonation of
Ascaris ova. When sludge samples were
              mixed mechanically by magnetic stirrers,
              85% to 90% of the ova were physically
              destroyed. Those ova that were recovered
              showed gross abnormalities such as vac-
              uolation,  cracked  egg  coats,  and
              granulation.
                Shaking the sludge sample on recipro-
              cating shakers during aerobic digestion
              resulted  in 32% embryonation  after 40
              days of shaking and  19% embryonation
              after 35 days. In only one experiment were
              52% of the recovered ova embryonated.
              No  ova were embryonated in one experi-
              ment that shook the sludge sample for 49
              days. Most of the nonembryonated ova in
              this trial  were vacuolated, showed  in-
              creased granulation, and were physically
 distorted. The percentages of unfertilized
 ova present in shaken sludge samples de-
 creased within  10 to 14 days. After  10
 days of shaking in one trial, 40% to 60%
 of the  initially seeded ova were arrested
 in the 2-, 4-, and 8-cell stages. This result
 indicated that embryonation had been initi-
 ated but stopped  early in  the cleavage
 stage of development.
   Aeration of aerobic sludge using an  air
 pump produced the highest percentages
 of ova embryonation. After 30 to 35 days
 of aeration, 62% to 68% of the ova were
 embryonated in sludges. The percentages
 of nonembryonated ova consistently de-
 creasesd over a 30- to 42-day period  of
 aeration. Increasing the detention time of
 digestion up  to 42 days produced little
 change in the percentage of  the embry-
 onation of the Ascaris ova.  Embryonated
 ova recovered from  aerated  sludge di-
 gesters showed no distortion, vacuolation,
 or granulation.
   The infectivity of ova recovered from
 sludge  aerated either by shaking or with
 air pumps was examined in two trials.  In
 each trial, rats were dosed with ova from
 sludge,  embryonated ova from hog feces
 (positive control), and physiological saline
 containing no ova (negative control). In the
 first trial, an average of 9% (11% and 8%)
 of the ova recovered from shaken  sludge
 were infective to rats, as demonstrated by
 the recovery of third-stage larvae from rat
 lungs. An average of 93% (95% and 91%)
 of the embryonated positive controls and
 no larvae from the negative controls were
 recovered. In the second trial,  an average
 of 12%  (10% and 14%) of the ova recover-
 ed from sludge aerated by air pumps were
 infective.  Postivie  controls showed  an
 average infectivity of  79%  (83% and
 75%), and no larvae were demonstrated
 in the lungs of negatively controlled rats.
 Recovered  larvae in all experiments ap-
 peared  to be normal and active.
  Ascaris ova are highly resistant to var-
 ious acids, alkalies, and corrosive  chem-
 icals. The inner, lipoid, vitelline membrane
 is primarily responsible for this effect. Ova
 surrounded only by  these membranes are
 resistant to these chemicals. The primary
 lipid component of the vitelline membrane
 is a waxy alcohol known as ascaryl alco-
 hol. Ascaryl alcohol appears to be a mix-
ture  of three closely related compounds —
ascosides A, B, and C. The lipid nature of
this  membrane makes it susceptible to
organic  solvents, surface active agents,
and noxious gases.  Possibly, the nonem-
bryonation and physical deformities of ova
may be  caused by  various chemicals or
gases that are generated during aerobic

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digestion  and that destroy  or  alter the
physiological  integrity of  this  vitelline
membrane. Once the membrane is altered,
the primary protective barrier is breached,
and the developing embryo is unprotected.
Alterations occur at this  point.

Field  Studies
  A number of human parasites were de-
monstrated in locally collected, undigested
sludge and sludge obtained from aerobic
and anaerobic digesters. Four human para-
sites were recovered from sludge: Ascaris
lumbricoides,  Trichuris  trichiura, Enter-
obius vermicularis, and hookworm. These
are the most common of the intestinal hel-
minth  parasites of man. The most  com-
mon parasite present in sludges from the
four collection sites was the common pin
worm or seat worm, E. vermicularis. The
second most common parasite  was the
large  intestinal  roundworm,  A. lumbri-
coides. Hookworm and the whip worm, T.
trichiura, were also present, but to a small
extent. Sludge samples from treatment
plants in Tallahassee, Florida, showed the
highest incidence of indigenous parasite
ova, followed (in order) by the Main Street,
Kanapagha, and University of Florida treat-
ment plants in Gainesville, Florida. Parasite
ova were  recovered from all sludges, re-
gardless of their treatment.
  An unexpected observation during ex-
amination of the sludge samples was the
high incidence of the  intestinal  helminth
E. vermicularis in Tallahassee, Florida. This
parasite has a wide geographic  range in
the United States. Cheng reported  an in-
fection rate of 32.9% in the American
population. Enterobius is the most com-
mon  helminth infection  in the United
States, sometimes reaching infection rates
of 60% in children and adults in various
institutions in Florida. An infection rate of
26.8% was reported among students of
five elementary schools in  and around
Tallahassee, Florida, These various studies
suggest that Tallahassee, Florida,  is an
endemic loci of enterobiasis in the state
of Florida.
  Ascaris  and  Trichuris are the most com-
mon and cosmopolitan helminth infections
in the world. The prevalence of these in-
fections in the population of North Amer-
ica  has been estimated at 4 million for
Ascaris and 2.2 million for Trichuris. These
two infections are still extremely common
in the rural areas of the southern United
States. With the prevalence of these infec-
tions in the population and with the fecun-
dity of these parasites (Ascaris produces
200,000 ova/day per female, and  Trichuris
produces  1,000 to 46,000  ova/day per
female),  it is  not  surprising that  these
parasitic ova were  found in locally col-
lected  sewage sludge.

Conclusions
  Indicator and pathogenic bacteria were
inactivated during  aerobic  digestion  of
sludge under laboratory conditions.  Proto-
zoans  and other predators were the pri-
mary agents responsible for inactivating
bacteria. Bacterial densities in undigested
sludge are reduced during aerobic treat-
ment of sludge under field conditions.
  The addition of fresh, undigested sludge
to aerobic digesters results in contamina-
tion  of the digested  sludge with undi-
gested sludge and thus diminishes the ef-
fectiveness of the  process  for reducing
bacterial densities.
  Enteric viruses are also inactivated dur-
ing the aerobic digestion of sludge under
laboratory conditions.  In contrast with
bacteria, the presence or absence of pre-
dators  had little influence on the inactiva-
tion  of viruses.  Aerobic treatment  of
sludge under field conditions reduces the
density of enteric  viruses.  As with bac-
teria, contamination of digested sludge
with fresh, undigested sludge leads to the
presence of viruses in the digested sludge.
  Aeration of  Ascaris  suum ova  in  the
presence  of 0.1 N  H2SO4 contamination
with fungi led to embryonation  of more
than 90% of the ova. Approximately 80%
of these ova were infective for rats. In con-
trast,  aeration in  aerobically  digested
sludge resulted in embryonation of 50%
or fewer of the ova. Only some 10% of
these ova  were  infective for rats. Thus
aerobic treatment of sludge reduces the
ability of ova in the sludge to embryonate
and reduces the infectivity of those ova
that  are embryonated. The relatively low
numbers of parasitic ova in undigested and
aerobically digested sludge from treatment
plants  made it difficult to determine the
effects of aerobic  sludge treatment on
these ova under field conditions.

Recommendations
  The  presence of bacterial pathogens,
enteric viruses, and parasitic ova in aero-
bically digested sludge suggests two ma-
jor areas of future research: (1) the fate of
these microbes during  subsequent treat-
ment or after  disposal (including during
sludge drying and application to land), and
(2) modif^ition  of  existing  sludge treat-
ment processes to reduce pathogens fur-
ther. One modification of sludge treatment
that  should be considered is the aeration
of sludge in batches without addition of
fresh,  undigested  sludge. All sludge re-
moved from  digesters operated  in this
manner would  be treated for the same
length of time and would not be contam-
inated with the bacterial pathogens, enter-
ic viruses, and parasitic ova that are often
found in undigested sludge. The possibility
of raising the temperature of sludge diges-
tion should also be considered.
  Additional studies are needed on the fate
of parasitic ova during aerobic sludge di-
gestion. Additional laboratory studies are
required to confirm our findings on the ef-
fects  of aerobic sludge digestion on the
embryonation and infectivity of Ascaris
suum ova. Larger volumes of sludge from
treatment plants should be processed to
provide the large numbers of ova needed
for embryonation and infectivity  studies.
  The full report was submitted in fulfill-
ment of Grant No. R806290 by the Univer-
sity of Florida under the sponsorship of the
U.S. Environmental Protection Agency.
                                                                                    .S. GOVERNMENT PRINTING OFFICE: 1986/646-116/20852

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      Samuel R. Far rah, Gabriel Bitton, and Stephen G Zam are with the University of
        Florida, Gainesville, FL 32611.
      B. V. Salotto was the EPA Project Officer (see below for present contact).
      The complete report, entitled "Inactivation of Enteric Pathogens During Aerobic
        Digestion of Waste water Sludge, "(Order No, PB 86-183 084/A S; Cost: $11.95,
        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, contact Joseph B. Farrell 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 45268
     BULK RATE
POSTAGE & FEES PAID
        EPA
   PERMIT No. G-35
Official Business
Penalty for Private Use $300
EPA/600/S2-86/047

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