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8) Pergamon Wal Sci. Ttch. Vol. 36, No. II. pp. 203-210, 1997.

@ 1997 IAWQ. Published by Elsevier Science Ltd


Primed in Great Britain.
0273-1223/97 $17'00 + 0'00
PH: S0273-1223(97)00684-7

REDUCTION OF FECAL
STREPTOCOCCUS AND SALMONELLA
BY SELECTED TREATMENT METHODS
FOR SLUDGE AND ORGANIC WASTE

Svend-Erik Jepsen,* Michael Krause**


and Henrik Grtittner***

*VKI, Agern Alii II, DK-2970 Hprsholm, Denmark


**The Danish Plant Directorate, Skovbrynet 20. DK-2800 Lyngby, Denmark
***Sophus Berendsen MS. Klausdalsbrovej J, DK2860 Sborg, Denmark

ABSTRACT

The increasing utilization of waste water sludge and source-separated organic household waste in agriculture
has brought the quality aspects into focus, among others the hygienic aspects. In this study, the reducting
effect on Fecal Streptococcus (FS) and Salmonella of different methods for stabilization and methods for
further treatment of sludge and organic waste has been investigated. The most common methods for
stabilization, i.e. aerobic and anaerobic stabilization, only reduce the indicator organisms by approximately I
logarithmic decade. Methods for further treatment of sludge and organic waste have shown reductions of
microorganisms allowing for unrestricted utilization in agriculture, meeting the product control: FS below
IOO/g and no Salmonella detected.
The effect of storage of sludge at summer and winter temperatures respectively has been investigated. At
temperatures (around 20'C) significant reductions of Salmonella were found, while the die out at low
temperatures (below W'C) was limited. FS was not reduced systematically during storage, and therefore, FS
is not usable as indicator organism for the hygienic properties of sludge during storage. 1997 IAWQ.
Published by Elsevier Science LId

KEYWORDS

Fecal Streptococcus; hygienic effect; Salmonella; sludge; source-separated organic waste; stabilization.

INTRODUCTION

The hygienic properties of sludge and organic waste is an important part of the handling and disposal.
especially when the sludge and organic waste are used for agricultural applications. The EEC directive
(EEC, 1986) on utilization of organic waste in agriculture operates with stabilization combined with area
restrictions. Stabilized sludge is expected to be non-odorous and to have significantly reduced pathogen
content. In Great Britain and USA a significant reduction is defined as a reduction of one log unit (Pike.
1991) and in most cases FS is used as indicator organism.

203
204 S.-E. JEPSEN et al.

The existing regulation of the use of sludge and source-separated organic waste in agriculture in Denmark
combines the treatment method with area restrictions for the land where sludge is applied. The statutory
order regulating the utilization of sludge and organic waste distinguishes between stabilization and sanitation
(Danish EPA, 1996). Sanitation is defined as heat treatment at 70'C for 1 hour or similar, combined with a
product control; no detectable Salmonella and below 1()() FS/g slUdge. The definition of stabilization is that
the sludge is biologically stable and non-odorous, but no available method exists to control this.

In Denmark, the increasing use in agriculture of waste water slUdge and source-separated organic household
waste has brought the hygienic aspects into focus. Most of the Danish biowaste is stabilized but not
specifically treated for pathogen reduction. Consequently, the Danish EPA has initiated a study of the
hygienic effects of waste stabilization methods common in Denmark where the fate of different indicator
organisms and pathogens are evaluated for selected treatment methods.

The most common methods for sludge stabilization in Denmark are aerobic stabilization (around 41 % in
1995) and mesophilic anaerobic stabilization (approximately 32% in 1995). Of the sludge reused in
agriculture, 36% is anaerobic stabilized, 50% is aerobic stabilized and 10% is lime stabilized. During the last
year the statutory order regulating the utilization of sludge and organic waste (Danish EPA, 1996) has been
revised, and limit values for four organic micropollutants have been included in the statutory order. These
new limits are expected to reduce the utilization of sludge from the present high reuse degree of around 68%
of the total sludge production in 1995. A majority of the waste water treatment plants in Denmark are built
with nitrogen and phosphorus removal with extended aeration and therefore, the sludge is aerobic stabilized.
Pan of the plants operate with primary settling or pre-precipitation followed by a biological stage with
nitrogen removal. The primary sludge is normally mesophilic anaerobic stabilized.

The amount of source-separated organic waste will increase significantly dUring the next years due to
attempts to minimize the waste deposit to landfills. In general, source-separated organic waste is treated by
anaerobic digestion or composting before agricultural reuse.

The effect of various treatment methods has been investigated in other countries (Strauch, 1988; Bruce et al.,
1990) but only limited knOWledge exists on the situation in Denmark. Therefore, the aim of this
investigation has been to screen the hygienic effect of the stabilization methods and a number of selected
methods for further reduction of pathogens. Beside this, the effect of storage of sludge has been investigated.
It has been shown from surveys in other countries that storage will reduce the content of indicator organisms
(WerkerIe, 1985). Ahmed and Sorensen (1995) have investigated the effect of storage in the laboratory at
controlled temperatures from 5'C to 50'C and they report decay rates among others for Salmonella. 90%
reduction (T90) at 5'C is found to 20 days, and T90 at 20'C is found to 6.4 days.

In this study, the sludge and organic waste were tested for the content of FS and Salmonella. FS was chosen
because it is more resistant in the environment than E. coli and most pathogens, and it is therefore
considered the best indicator organism in organic waste. Salmonella was chosen as the most important
pathogen in organic waste.

MATERIALS AND METHODS

Treatment plants investi~ated

The waste water treatment plants and composting plants included in the survey are listed in Table 1.

Sampling methods

All samples from the different treatment methods were collected as grab samples, typically mixed of a
number of sub-samples. The samples were kept refrigerated and analysed within 24 hours.
Reduction of Fecal Streptococcus and Salmonella 205

Two samples per day were collected at each sampling point and double analysis was performed at each
sample. The logarithmic average of these measurements was used in the calculation. For all treatment plants
samples were collected on October 94, February 9S and June 9S in order to evaluate seasonal changes in
performance and content of microorganisms.

Table I. Treatment plants investigated and methods used for stabilization/sanitation

Aerobic Anaerobic Lime High Composting


addition temperature

Skrevinge WWTP A
Hillered WWTP A A
Ejby Melle WWTP A A
Marseliborg WWTP A A
Haderslev WWTP A A
StruerWWTP A
Kestrup Composting Plant B
KertemindelMunkebo WWTP C

A = sludge; B = source-separated organic household waste;


C = sludge and source-separated organic household waste

Analytical methods

Fecal Streptococcus (FS): The content ofFS was determined by sowing the sample and decimal dilutions of
it on Bacto Enteroccus Agar. The characteristic red colonies were counted after incubation at 37"C for 48
hours. A suitable number of colonies were verified by microscopy. by the catalase test, and by sowing the
sample on Bacto Bile Esculin Azide Agar.

Salmonella: The Salmonella content was analysed by the MPN (Most Probable Number) method according
to ISOIDIS 6340/1986.

Total dry solids at IOS'C and total volatile dry solids ignited at SSO'C were analysed according to Standard
Methods ( 1989).

The effects of storage/time were studied by storing S m 3 of anaerobic stabilized sludge and S m 3 of aerobic
stabilized sludge in two containers. The content of FS and Salmonella was followed for up to six months.
Samples were taken as vertical profiles. excluding the top 20 cm of sludge. The samples were intensively
mixed before a sub-sample was taken for analysis. The effects of storage in a Danish climate were evaluated
during summer temperatures (20-30'C in the sludge) as well as during winter temperatures (O-W'C in the
sludge). The anaerobic stabilized sludge had approximately 28% dry solids content and the aerobic
stabilized sludge had approximately 20% dry solids content due to the differences in dewaterability.

RESULTS AND DISCUSSION

Anaerobic stabj!jzatjon

The survey included three waste water treatment plants with anaerobic digestion in the mesophilic range.
Figure I shows the logarithmic average of the content in the outlet from the anaerobic process. Salmonella
was only measured at Hillered WWTP. Large variations in the measurements occurred especially at the inlet
206 S.-E. JEPSEN el al.

to the anaerobic digesters. The variations from day to day within the same plant were found to be on a
comparable level from plant to plant.

The reduction of FS in the mesophilic digestion varies within each individual treatment plant as well as from
one plant to another. In general, a reduction of 1-1.5 decades was measured. as can be seen in Fig. 2. The
measurements in the inlet is a grab sample from the same day as the measurement taken in the outlet. The
typical retention time for solids is 20-30 days and consequently the reductions presented in Fig. 2 only give
the magnitude of the reduction. The content of Salmonella found is typically between 500 and 5.000
SalmonellallOO g sludge in the anaerobic stabilized sludge with a reduction similar to the reduction of FS.

10.000.
x
! 0Cl"

0

I .
0 +

. * 0Cl"

1.000.000

OJ

I FebK
0

~
I FebK
x
~ "
0
+ I I ..
J... K
~
~
I .
I J... K
"---
~ )C

100,000 I

SIlIOVinge .-.red

-~
_g --- Ejby __ -..otg Ejby

--
Figure I. Content and variation of FS in aerobic and anaerobic stabilized sludge at different waste water treatment
plants.

O ctD4

O ct 94
~
Feb 95
ffi!I

1 ~
Feb 95

... Jun95
~
j Jun 95
g
! o.~
<I)
IL

04---

-O.~-+---------.....-------r------------1
HilI.,od Marsen.borg Ejby Mello

Fi,ure 2. Anaerobic stabilization. decades reduction of FS al differenltimcs of the year, II =ne,ative reduction due
10 ,rab s.mphn. althe same day.
Reduction of Fecal Streptococcus and Salmonella 207

Aerobic stabi!izaljon

In Denmark, sludge from. waste water treatment plants with extended aeration for nitrogen removal is
normally considered as aerobic stabilized. The sludge age in these plants varies between 20 and 60 days,
with approximately 60% aerobic sludge age.

Four different treatment plants with aerobic stabilization were examined for the content of FS and at two of
the plants also for the content of Salmonella. One plant operates without presettling (Ska:vinge WWfP),
while the others operate with presettling or pre-precipitation. The sludge age varies from 25 days at Hillerlild
WWfP to more than 60 days at Ska:vinge WWfP. As seen from Fig. I, these differences do not influence
the content of FS per gram of dry solids in the stabilized sludge. It can be seen that the variation from day to
day is as large as the variations from plant to plant The Salmonella content is typically between 10,000 and
1,000,000 Salmonella/I 00 g dry solids in the stabilized sludge.

In Fig. I aerobic and anaerobic stabilization are compared based on dry solids. FS is around I decade lower
in anaerobic sludge than in aerobic sludge. Similarly, the Salmonella content is lower in anaerobic stabilized
sludge, but still a significant content is found.

Methods for further reductjon of patho~ens

Three methods for further reduction of pathogens are monitored: Lime stabilization, aerobic thermophile
treatment before anaerobic mesophile stabilization (the UTB-process) and composting. At lime stabilization
the pH is raised to above 12.3 with CaO and during rehydration of the lime the temperature will raise as
well. Indicator organisms are measured 24 hours after lime addition. The UTB-process operates at a
minimum temperature of 62'C for at least 70 minutes. The average retention time is 1.9 days. The two
composting facilities investigated were windrow composting facilities. The temperatures in the windrows
were controlled and they were turned regularly.

As can be seen in Table 2, all three methods result in a product with low bacterial content. Lime as well as
temperature treatment result in bacterial numbers below the detection limit. In the two composting facilities
FS can vary from below 100 to 1,000 FS/g. The initial content in the organic waste from the composting
plants is significantly higher than in the sludge.

Of the three methods only lime stabilization is approved as sanitation according to the Danish statutory order
(Danish EPA, 1996). In the UTB-process, the temperature is 62'C. It should be raised to 70'C in order to
comply with the regulations. In the windrow composting plants the problem is to secure that all parts of the
waste are treated at sufficiently high temperatures.

Table 2. Hygienic effects of temperature, lime and composting

Before treatment (avg.) After treatment (avg.) Reduction


FS/g Sa1./100 g FS/g Sa1./100 g

Temperature 70,000 3,300 < 100 <2 >3


(UTB-process)
Lime addition 200,000 300 < 100 <2 >3
Composting 4,000,000 3,600 100 <2 >4

FS = Fecal Streptococcus; Sal. = Salmonella


= Decades reduction of Fecal Streptococcus
208 S.E. JEPSEN ., al.

Stora~e of slud~e

Figure 3 shows the FS con!ent during storage of aerobic stabilized sludge at summer temperatures between
20'C and 30'C and at winter temperatures between O'C and WOC. The temperature was measured in the
centre of the sludge container and showed only small variations. At summer temperatures. aerobic stabilized
sludge showed reductions of FS from 1.000.000 FS/g to approximately 10.000 FS/g within two months.
During the last month no further reduction was found (see Fig. 3). At the high temperature storage
Salmonella was reduced to below the detection limit within one month. At low temperatures the reduction of
FS was less than I decade. and even within 6 months Salmonella was not reduced to below the detection
limit. see Fig. 4.

---
Summ.. 85

Surnm.. 84
-&-
wont..

o ~ ~ eo 80 100 I~ 1~ 180 180


Days

Figure 3. Effect on FS content in storage of aerobic stabilized sludge.

-- ..........
_
...-ole_

-
-&-

anaerobic aummw

10 20 40 eo 10 100 120 140 110 180


Oaya

Figure 4. Effect on Salmonella content in storage of aerobic and anaerobic sludge.

Figure S shows the FS content in anaerobic stabilized sludge at summer as well as winter temperatures. At
high temperatures. anaerobic stabilized sludge showed a reduction of FS from 20.000 FS/g to approximately
20 FS/g within four months. Before storage. the content of Salmonella in the sludge was approximately
10.000 Saimonella/IOOg in the winter sample and around 300 Salmonella/IOOg in the summer sample. At
the high temperature storage. Salmonella was reduced to below the detection limit within one month. At low
Reduction of Fecal Streptococcus and Salmonella 209

temperatures Salmonella was still detected at the detection limit of I Salmonella/IOO g after six months, see
Fig. 4. The content of FS was reduced by I to 2 decades, but the reduction was not stable with time.

The decay rates reported by Ahmed and Sorensen (199S) and the decay curve for anaerobic stabilized sludge
given in Fig. 4 are similar for the S'C decay rate at winter temperatures and for the 20'C decay rate at
summer temperatures.
1.000.000
-+-

........
Summer 85

Summer 84
........
Wint

.
J
t il

1.000
~
"-

100

10

1+--"'T""-.......
o ~ ~
---.----.---,r---..--.........- ........- - l
~ ~ 100 1~ 1~ 1~ 1M
Day.
Figure 5. Effect on FS content in storage of anaerobic stabilized sludge.

CONCLUSIONS

It can be concluded that the most common methods for sludge treatment in Denmark will not reduce the
pathogens and indicator organisms to a level which is acceptable for unrestricted utilization of the sludge in
agriculture. For Salmonella, the typical reduction found is around I decade, which is in accordance with the
rules in the EEC directive for stabilized sludge (EEC directive, 1986). No seasonal variations in the hygienic
properties of the sludge and organic waste were found.

Furthermore, the study confirms the hygienic effect of the investigated methods for further treatment of
sludge and source-separated organic waste (aerobic thermic treatment, quick lime treatment and
composting), where it has been found that high temperature as well as high pH will inactivate the
microorganisms.

The very significant effect of storage, especially on Salmonella reduction at temperatures of 20'C and
higher, may improve the hygienic properties of the sludge utilized in agriculture. At temperatures below
IO'C only minor effects are seen even after long periods of storage.

REFERENCES

Ahmed. A. U. and Sorensen. D. L. {I 995). Kinetics of pathogen destruction during storage of dewatered biosolids. Water
Environment Reuarch. 67, 143-150.
Bruce, A. M. Pike. E. B. and Fisher. W. J. (1990). A review of treatment process options to meet the EC sludge directive. J.
lWEM,4.
Danish EPA (1996). Statutory order no. 823. September 1996. on application of sludge. sewage and compost etc. for agricultural
purpose (in Danish).
EEC directive, June 12. 1986. (861278IEEC).
ISOIDIS 6340 (1986). Microbiology general guidance on methods for the detection of Salmonella.
Pike. E. B. (l99\). Treatment and Use of Sewage Sludge and Liquid Agricultural Wastes. P. L'Hermite (cd.). ElseVier Applied
Science. London.
210 S.-E. JEPSEN et al.

Standard Methods (1989). Standard methods for the examination of water and waste water. 17th edition. EP. by L. S. Clesceri. A.
E. Greenberg and R. R. Trussell.
Strauch, D. (1988). Hygienic Aspects of the Treatment and Use of Organic Sludge and Liquid Agricultural Waste. D. Strauch, H.
Over and P. L'Hermite (eds.). Elsevier Applied Science, London.
Werkerle, J. (1985). Epidemiological Studies of Risks Associated with Agricultural Use of Sewage Sludge: Knowledge and Needs.
J. C. Block, A. H. Have1ar and P. L'Hennite (eds). Elsevier Applied Science, London.

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