Bacteriophages as model organisms for monitoring the
microbiological quality of the effluent of a wastewater
treatment plant
MANDILARA GD*, MAVRIDOU A**, LAMBIRI M*, VATOPOULOS A*,
RIGAS F***
*Department of Microbiology
National School of Public Health
196, Alexandras Av.
115 21 Athens GREECE
**Technological Educational Institution of Athens
Department of Medical Laboratories
Ag.Spyridonos
122 10 Aigaleo GREECE
*** School of Chemical Engineering
National Technical University of Athens
9, Iroon Polytechniou Str.
157 00 Athens GREECE
Abstract: - The discharge of treated wastewater in surface water and its reuse in agriculture
requires a good microbiological quality of the effluent, in order to protect the environment
and the public health. The use of bacterial indicators for monitoring the microbiological
quality of the effluent is not always adequate, since pathogens, such as enteroviruses, are
more resistant to natural inactivation and to treatment processes. Therefore, the use of
bacteriophages as indicators has been studied recently. In this study an effort was made to
correlate the number of bacterial indicators with the presence of three groups of
bacteriophages; somatic coliphages, F-RNA specific phages and phages of Bacteroides
fragilis, in raw and treated sewage and septage. Samples of each treatment stage of two
sewage treatment plants in Athens, in a 2-year period, were collected monthly, and analyzed
for total coliforms, E.coli, intestinal enterococci and the three groups of bacteriophages. A
clear correlation between the number of bacterial indicators and the presence of
bacteriophages was observed. E.coli and enterococci concentrations of 103 cfus/100ml
consist a threshold for the presence of somatic coliphages in wastewater. E.coli
concentrations of 106 cfus/100ml and enterococci of 105 cfus/100ml consist a threshold for
the presence of F-specific bacteriophages, and E.coli and enterococci concentrations of 106
cfus/100ml consist a threshold for the presence of B.fragilis phages.
Key words: - Bacteriophages, indicators, sewage, wastewater, treatment, reuse.
1 Introduction
The reuse of treated wastewater in
agriculture and aquaculture and the
conditions of the effluent discharge in
surface waters are rapid developing fields.
Among pathogens shed in human waste
through faeces are bacteria, viruses and
protozoa [1]. Legislation [2] and
guidelines
[3]
indicate
the
microbiological quality of the treated
wastewater to be re-used in agriculture
and the quality of the surface waters that
receive the effluents of a sewage plant
[4].
2 Microbiological quality of
treated wastewater
1
2.1 Bacterial indicators
The isolation and identification of
pathogenic microorganisms demand
complicated, expensive, and time
consuming methods. Therefore, the use of
indicators for the assessment of water
quality and for the effectiveness of a
treatment process has been established.
Ideally, an indicator should have the same
ecology as the pathogens to which it
relates, should be detectable by simple,
rapid and inexpensive methods and
should present similar to slightly greater
resistance to natural inactivation and
treatment processes than that of surrogate
microorgranisms.
Several studies have demonstrated that
the current microbiological indicators of
water quality, such as total coliforms,
E.coli, and intestinal enterococci are
inadequate to indicate the presence of
enteroviruses. Enteroviruses are known to
be more resistant to natural inactivation
and to water and wastewater disinfection
than the current bacterial indicators of
water quality. [5], [6], [7].
2.2 Bacteriophages
Bacteriophages are of similar structure,
composition, morphology and size as
enteroviruses and are also found in
sewage. Moreover, the methods for
enumeration of bacteriophages are very
simple, inexpensive and require no
confirmation. Much research has been
carried out on the use of bacteriophages
as indicators for the presence of
enteroviruses in water and as indicators
for the efficiency of a treatment process
[7], [8], [9], [10], [11], [12], [13], [14].
Potential new model organisms include
somatic coliphages [15], [16], [17], Fspecific bacteriophages [17], [18] and
phages infecting Bact. fragilis [8], [17],
[19], [20].
supplementary indicators of faecal
pollution
for
monitoring
the
microbiological quality of treated
wastewater.
3 Materials and Methods
3.1 Wastewater treatment plants.
Sewage samples were collected from two
sewage treatment plants of Athens. The
first one (A) deals with the urban sewage
of the city and it includes a pretreatment
stage and primary settling.
The other plant (B) treats part of the
urban sewage and all the septages of the
city of Athens. This plant includes a
pretreatment stage separate primary
settling for the urban sewage and the
septages, biological treatment, secondary
settling and a disinfection stage of
uncertain effectiveness, since the mean
concentration of residual chlorine was 0.3
ppm.
BOD, COD and concentrations of
bacterial indicators of raw wastewater are
given in Table 1.
3.2 Sampling
Samples were collected monthly, over a
2-year period (from November 2000 to
September 2002), from each of the
following treatment units: raw sewage
after the pretreatment unit at plant A
(A1), outlet of the primary settling tank
(A2), raw urban sewage after the
pretreatment unit at plant B (B1), raw
septages at plant B (B2), outlet of the
primary settling tank (of the urban sewage
B3, and of the septages B4) , inlet of the
biological treatment unit (B5) and outlets
of the biological treatment unit (B6),
secondary settling tank (B7) and
disinfection unit (B8).
3.3 Bacterial indicator analysis.
2.3 Aim of the Research
The aim of the present research is to study
whether there is a correlation between the
concentration of the bacterial indicators
and the bacteriophages in raw and treated
wastewater and evaluate their use as
The enumeration of the indicator bacteria
was made using the membrane filtration
method [21].
2
Table 1. Characteristics of raw wastewater at the two treatment plants. (BOD: Biochemical
Oxygen Demand, COD: Chemical Oxygen Demand, TC: total coliforms, EC: E.coli, IE:
intestinal enterococci)
BOD (mg/l)
COD (mg/ml)
TC (cfus/100ml)
Plant A
raw
wastewater
378
837
1.9x108
raw
wastewater
354
659
1.0 x108
EC (cfus/100ml)
IE (cfus/100ml)
1.0x107
4.0x106
4.5x106
2.5x106
3.4 Bacteriophage analysis
All phages were quantified by the double
– agar – layer method according to
standard procedures [22], [23], [24].
3.5 Quality assurance
A first-line quality control was
performed using reference materials, for
both bacteria and bacteriophages.
4 Results
Table 2 and Table 3 present the
relationship between the concentration of
E.coli
and
intestinal
enterococci
respectively, and the presence of the three
groups of bacteriophages in raw and
treated sewage and septage. In samples
where the concentration of E.coli was 105
cfus/100ml, SOMCPH were detected at
100% of the samples, FRNAPH at 55%
and BFRPH were isolated in none of the
samples.
In samples of E. coli
concentration ≥ 106 cfus/100ml all three
groups of phages were detected in almost
all samples. Moreover, in samples of
enterococci
concentration
of
105
cfus/100ml, SOMCPH were detected at
100% of the samples, FRNAPH at 83.3 %
and BFRPH at 63.9% of the samples. In
samples of enterococci concentration ≥
106 cfus/100ml, SOMCPH and FRNAPH
were detected at 100% of the samples.
BFRPH were detected at 79.6% of
Plant B
raw
septages
1116
2180
7.4x107
4.0x106
8.5x105
samples of enterococci concentration 106
cfus/100ml and at 100% of samples of
enterococci
concentration
≥107
cfus/100ml.
5 Conclusion
In this study, an effort has been made to
determine a threshold for the presence of
bacteriophages according to their
detection in at least 80% of the sewage
samples with a given concentration of
bacterial indicators. A clear correlation
between the concentration of E.coli and
enterococci with the presence of the three
groups of bacteriophages in raw and
treated sewage has been detected. E.coli
and enterococci concentrations of 103
cfus/100ml consist a threshold for the
presence of somatic coliphages in
wastewater.
Moreover,
E.coli
concentrations of 106 cfus/100ml and
enterococci of 105 cfus/100ml consist a
threshold for the presence of F-specific
bacteriophages,
and
E.coli
and
enterococci concentrations of 106
cfus/100ml consist a threshold for the
presence of B.fragilis phages.
The revised WHO guidelines for the
microbiological quality of treated
wastewater used in agriculture and
aquaculture [3], [25] indicate for
unrestricted irrigation (that is, for uses
that include crops likely to be eaten raw)
3
≤ 103 faecal coliforms in 100 ml; for
irrigation of commercially processed and
fodder crops (restricted irrigation) the
guideline limit is ≤ 105 faecal coliforms
/100 ml using the spray or sprinkler
irrigation technique; and ≤ 103 faecal
coliforms /100 ml for flood or furrow
technique.
Likewise, WHO recommends a tentative
bacterial guideline of ≤ 103 faecal
coliforms /100 ml for the treated
wastewater used in aquaculture. These
guidelines could be supplemented with
the use of bacteriophages. For example,
according to the findings arising from the
present research, “no detection of somatic
coliphages” could be used as a limit in the
case of unrestricted irrigation and
aquaculture, and “no detection of Fspecific phages and phages of B. fragilis”
in the case of restricted irrigation.
The legislation for the quality of the
treated wastewater discharged in surface
waters [4] does not provide specific limits
regarding the microbiological quality of
the effluent. However, it is very important
to monitor the quality of the discharged
treated effluent in order to protect the
microbiological quality of the receiving
waters. Bacteriophages may be a valuable
tool for achieving improvements in this
field. Their advantage vis-à-vis faecal
indicators is that the methods for the
detection
and
enumeration
of
bacteriophages produce results in just 6
hours without requiring any confirmation
step. Moreover, they can be used as
model/surrogates for enteric viruses since
they closely meet basic requirements for
this function [26], [27].
Table 2: Relationship between the concentration of E.coli and the presence of the three
groups of bacteriophages in raw and treated sewage. (SOMCPH: somatic coliphages,
FRNAPH: F-RNA specific phages, BFRPH: phages of Bacteroides fragilis.)
E.coli (cfus/100ml)
3
4
10 -10
105
106
107
No. of
samples
35
20
92
33
SOMCPH
94.3
100.0
100.0
100.0
% presence of:
FRNAPH
5.7
55.0
96.7
97.0
BFRPH
0
0
82.6
94.0
Table 3: Relationship between the concentration of intestinal enterococci and the presence
of the three groups of bacteriophages in raw and treated sewage. (SOMCPH: somatic
coliphages, FRNAPH: F-RNA specific phages, BFRPH: phages of Bacteroides fragilis.)
Intestinal
enterococci
(cfus/100ml)
103-104
105
106
≥107
No. of
samples
SOMCPH
39
36
98
7
92.3
100.0
100.0
100.0
% presence of:
FRNAPH
5.1
83.3
97.0
100.0
BFRPH
0
63.9
79.6
100.0
4
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