Italian Journal of Zoology

ISSN: 1125-0003 (Print) 1748-5851 (Online) Journal homepage: http://www.tandfonline.com/loi/tizo20

Protozoa in wastewater treatment processes: A

minireview
P. Madoni
To cite this article: P. Madoni (2011) Protozoa in wastewater treatment processes: A
minireview, Italian Journal of Zoology, 78:1, 3-11, DOI: 10.1080/11250000903373797
To link to this article: http://dx.doi.org/10.1080/11250000903373797

Copyright 2011 Unione Zoologica Italiana

Published online: 10 Dec 2010.

Submit your article to this journal

Article views: 4196

View related articles

Citing articles: 17 View citing articles

Full Terms & Conditions of access and use can be found at

http://www.tandfonline.com/action/journalInformation?journalCode=tizo20
Download by: [University of Engineering & Technology Taxila]

Date: 23 January 2017, At: 10:57

Italian Journal of Zoology, March 2011; 78(1): 311

REVIEW
TIZO

Protozoa in wastewater treatment processes: A minireview

P. MADONI*
Protozoa in wastewater treatment

Department of Environmental Sciences, University of Parma, Parma, Italy

(Received 18 June 2009; accepted 19 September 2009)

Abstract
Biological wastewater treatment is a process of increasing importance in a world with an ever-increasing human population.
Wastewater treatment facilities are designed to maintain the high density and activity levels of those microorganisms that
carry out the various purification processes. Protozoa are one of the most common components in these man-made ecosystems and play an important role in wastewater purification processes. Protozoa are responsible for improving the quality of
the effluent, maintaining the density of dispersed bacterial populations by predation. Studies of the relationships between
protozoa and physicochemical and operational parameters have revealed that the species structure of these communities is
an indicator of plant efficiency. The Sludge Biotic Index (SBI), an index based on the structure and abundance of the
microfauna inhabiting the activated sludge and mixed liquor, has been devised to monitor activated-sludge plant performance. Heavy metals and other pollutants are toxic to most microorganisms at certain concentrations. These toxicants are
common pollutants of sewage, particularly where there is industrial waste input. The protozoa community is a complex
assemblage of interacting organisms, often including species that are sensitive, resistant or intermediate in their tolerance to
pollutants. Many studies conducted on contaminated activated sludge and mixed liquor have revealed changes in the
dynamics of the protozoa community. Tests on the acute toxicity of pollutants on ciliates have revealed that these microorganisms are useful bioindicators for evaluating the toxicity of waters polluted by different concentrations of metals.

Keywords: Wastewater treatment, monitoring, community structure, protozoa, toxicants

Introduction
Biological wastewater treatment processes rely on
the natural self-purification ability of microbial communities. Nevertheless, they differ from aquatic
environments due to certain characteristics, such as
a strong flow of organic load into the system,
accelerated decomposition processes, short biomass
turnover time, and the prevalence of heterotrophic
organisms. Due to these characteristics, biological
wastewater treatment processes can be regarded as
man-made ecosystems subjected to extreme conditions (Antonietti et al. 1981), so that of all the heterotrophic organisms only protozoa and small metazoa
with life cycles shorter than the sludge retention
time are able to compete in these processes.

The presence of protozoa in biological wastewater treatment processes was observed almost as
soon as each process was introduced, but it has only
been in recent years that the significance of these
microorganisms has been underlined. The organisms
most directly involved in wastewater treatment are
the bacteria. They dominate all other groups, in
number and biomass, and affect the process of mineralization and elimination of organic and inorganic
nutrients. In modern systems, where there is a low
load and high sludge retention time, the presence of
protozoa such as ciliates, flagellates, and amoebae,
or even small metazoa, is very common. These
eukaryotic organisms are able to feed on particulates,
such as suspended bacteria. It is generally assumed
that their primary role in wastewater treatment is the
clarification of the effluent.

This paper is dedicated to Maria Umberta Delmonte Corrado for her contribution to the development of Italian Protistology.
*Correspondence: Paolo Madoni, Dipartimento di Scienze Ambientali, Universit di Parma, Via Usberti 11/A, 43100 Parma, Italy. Fax: +39 521 905402.
Email: paolo.madoni@unipr.it

ISSN 1125-0003 print/ISSN 1748-5851 online 2011 Unione Zoologica Italiana

DOI: 10.1080/11250000903373797

P. Madoni

Occurrence and diversity of protozoa

Percolating filter process
Lists of the protozoa found in percolating filters
have been given by many authors (Agersborg &
Hatfield 1929; Barker 1943; Curds & Cockburn
1970a). More than 200 species have been identified by these authors, and a complete list has been
reported by Curds (1975). Ciliophora is the phylum of protozoa that contributes the greatest
number of individuals to the microfauna of a percolating filter. Ciliates range from 500 to 10,000
individuals per ml of liquor (Curds & Cockburn
1970b). Certain species have been found in very
large numbers (Chilodonella uncinata, Vorticella convallaria, Opercularia microdiscum and Carchesium
polypinum). However, their frequency has not necessarily been associated with a dominant numerical
position in the population. One such species is
Cinetochilum margaritaceum, which, although
present in 54% of cases, has never been found in
large numbers (Curds & Cockburn 1970b). Other
species such as Acineria uncinata, Amphileptus pleurosigma and Vorticella microstoma, although frequently found, were usually present in small
numbers. In a study of the structure of a protozoan
community in a percolating filter (Mistri et al.
1994), large variations in the densities of some ciliate species were observed. The structure of the
protozoa community was characterized by the
high-density values of the crawling hypotrich
Aspidisca cicada associated with a rich assemblage
of attached peritrichs. Nevertheless, species blooms
were frequent and stalked ciliates often exhibited
large density oscillations. Sudden demographic
blooms of some ciliate species (mainly Dexiotricha
colpidiopsis, Uronema nigricans, Acineria uncinata,
Chilodonella uncinata, Paramecium caudatum and
Colpidium colpoda) were indicative of a less mature
condition.

Rotating biological contactor process

Protozoa are commonly present in rotating biological contactors (RBC), where they colonize the
biofilm. Lists of identified species appearing in
RBC systems have been given by certain authors
(Madoni 1981; Madoni & Ghetti 1981). Comparative studies of the occurrence (Madoni & Ghetti
1981; Kinner & Curds 1987; Chung & Strom
1991; Hul 1992; Prez-Uz et al. 1998) and distribution (Madoni et al. 1979; Luna-Pabello et al.
1992; Martin-Cereceda et al. 2002) of ciliate populations in RBC systems have also been made. A

relationship between the organic load and the

distribution of microorganisms in the reactor has
been found; heterotrophic flagellates and freeswimming ciliates such as Cyclidium glaucoma and
Colpidium colpoda are dominant in the early stages,
where the organic load is higher, whilst testate
amoebae and attached ciliates such as Epistylis
plicatilis and Zoothamnium procerius are dominant in
the final stages, characterized by low BOD5 values.
The quantitative importance of protozoa in RBC
systems has been determined in terms of biomass
(Madoni 1994a): a ciliate biomass value of 314 g
cm2 (dry weight) has been measured, corresponding to over 12% of the volatile solids on the RBC
biofilm.

Activated-sludge process
Since the installation of the first activated-sludge
treatment facilities in 1922, many authors have
noted the presence of free-living protozoa. These
microorganisms are commonly found in the mixed
liquor of activated-sludge plants and numbers of
the order of 320 106 cells l1 are often reported.
It has been estimated that the protozoa biomass
can reach values of 250 mg l1 (dry weight), constituting over 9% of the volatile solids (Madoni
1994a). Lists of the protozoa species found in activated-sludge plants have been reported by a
number of authors (Agersborg & Hatfield 1929;
Ardern & Lockett 1936; Clay 1964; Brown 1965;
Curds & Cockburn 1970a; Morisita 1970). A
complete list of 228 species of protozoa has been
reported by Curds (1975). Since wastewater treatment is a process of increasing importance in a
world with an ever-growing human population, the
presence and action of microbial communities has
received particular attention in recent years. Consequently, revised lists of protozoa have been made
(Madoni & Ghetti 1981; Augustin & Foissner
1992; Madoni et al. 1993; Foissner & Berger 1996;
Martin-Cereceda et al. 1996; Amann et al. 1998;
Ettl 2001; Madoni 2002; Chen et al. 2004). Of the
228 protozoa species listed for the activated-sludge
plants, about 160 belong to the phylum Ciliophora,
but only a limited number of these have been
observed frequently (Table I). Despite the fact that
the ciliate community of the activated sludge is
slightly different from that of the percolating filters,
some species, such as Acineria uncinata, Vorticella
convallaria, Vorticella microstoma, Opercularia
coarctata and Aspidisca cicada, are commonly
observed in both processes. In a well-functioning
activated-sludge plant, the protozoa community is

Protozoa in wastewater treatment

Table I. Occurrence of protozoa in activated sludge plants in
Italy (V, very common; C, common; F, frequent; R, rare).
Group
Large flagellates
Amoebae
Testate amoebae

Species

Frequency

Euglena sp.
Peranema sp.

F
F

Arcella sp.
Euglypha sp.
Centropyxis sp.

F
F
R

Bacterivorous ciliates
Free-swimming
Cinetochilum
margaritaceum
Colpidium colpoda
Cyclidium glaucoma
Dexiostoma campylum
Glaucoma scintillans
Paramecium aurelia
complex
Paramecium caudatum
Pseudocohnilembus
pusillus
Spirostomum teres
Uronema nigricans
Crawling
Acineria uncinata
Aspidisca cicada
Aspidisca lynceus
Chilodonella uncinata
Drepanomonas revoluta
Euplotes aediculatus
Euplotes affinis
Euplotes patella
Trithigmostoma
cucullulus
Trochilia minuta
Stylonychia sp.
Attached
Carchesium polypinum
Epistylis chrysemidis
Epistylis entzii
Epistylis plicatilis
Opercularia articulata
Opercularia coarctata
Opercularia nutans
Stentor coeruleus
Vaginicola ingenita
Vorticella aquadulcis complex
Vorticella convallaria complex
Vorticella infusionum complex
Vorticella microstoma complex
Vorticella octava complex
Zoothamnium arbuscula
Carnivorous ciliates
Acineta tuberosa
Amphileptus claparedei
Coleps hirtus
Litonotus crystallinus
Litonotus cygnus
Litonotus lamella
Plagiocampa rouxi
Podophrya sp.
Prorodon sp.
Spathidium sp.
Tokophrya lemnarum
Tokophrya quadripartita

F
R
R
R
R
R
R
R
F
R
V
V
C
F
F
F
F
R
F
C
R
C
C
F
V
F
F
R
R
F
V
V
R
R
C
F
C
F
F
C
R
F
V
R
C
R
F
C

dominated by peritrichs (Vorticella spp., Carchesium

spp., Zoothamnium spp., Epistylis spp.) and hypotrichs (Aspidisca spp., Euplotes spp.). In some cases,
the dominance of cyrtophorids (Chilodonella spp.,
Trochilia minuta) or testate amoebae (Arcella spp.,
Euglypha spp.) can be observed. In a study devoted
to reconstructing the species structure of the ciliate
community in activated-sludge processes (Madoni
& Ghetti 1981), it was found that of the 45 species
present in 39 different plants, five were recurrent
and representative of the principal community
(Aspidisca cicada, Vorticella convallaria, V. striata
octava, Epistylis plicatilis and Trochilia minuta).
Other species, such as Trithigmostoma cucullulus,
Euplotes sp. and Zoothamnium pygmaeum, have no
affinity with each other, but show affinity with
some species of the recurrent group, and can therefore be regarded as associated species.
The role of protozoa in wastewater treatment
Protozoa were originally thought to be harmful to
the activated-sludge process (Fairbrother &
Renshaw 1922). However, some authors (Curds et
al. 1968) were able to assess the role of these
organisms and to quantify the magnitude of their
effect upon the effluent quality. These authors
found that with no protozoa in the mixed liquor the
BOD5 of the effluent and other parameters (e.g.
the level of organic carbon and mixed liquor
suspended solids (MLSS)) were higher. The positive effects of protozoa on carbon mineralization by
bacteria in activated sludge are well known (Ratsak
et al. 1996). The excretion of mineral nutrients by
protozoa results in an accelerated usage of the carbon
source by the bacteria (Coleman et al. 1978; Bloem
et al. 1988; Tezuka 1990). Furthermore, protozoa
excrete growth-stimulating compounds that can
enhance bacterial activity (Nisbet 1984; Horan
1990). Nevertheless, these indirect effects of protozoa on bacterial growth cannot increase carbon
mineralization under carbon-limitation conditions
(Curds 1982). Thus, in wastewater systems with a
low substrate concentration, the process is of little
importance. Among the direct effects of protozoa
on bacteria, grazing plays an important role.
Protozoa, in fact, are considered to be the most
important bacterivorous grazers. The clearance
rates of several protozoa inhabiting activated sludge
range from 4 107 to 1 106 ml medium
protozoa1 h1 (Bloem et al. 1988, 1989). Even
though some protozoa (crawling ciliates and other
forms) can eat flocculated bacteria, most protozoa
(prevalently attached ciliates) can only graze on
suspended bacteria and particles; in this way they

P. Madoni

have a significant effect on the effluent quality. It is

generally assumed that the primary role of protozoa
in wastewater treatment is the clarification of the
effluent (Curds et al. 1968; Madoni 2003). In the
presence of ciliates, a reduction of the density of
viable Escherichia coli was also observed (Curds &
Fey 1969; Mallory et al. 1983). Several studies
have focused specifically on the role of protozoa
predation in nitrogen cycling in activated sludge
(Verhagen & Laanbroek 1991; Petropoulos &
Gilbride 2005; Pogue & Gilbride 2007). It has been
observed that the presence of protozoa increases the
per-cell nitrification rate, probably because of the
ability of protozoa to influence bacterial growth.
Protozoa release inorganic and organic products
into their surroundings. These products are mainly
recycled nutrients, such as nitrogen, phosphorous,
and organic carbon, but might also include stimulatory compounds that contribute to the dissolved
organic carbon pool and affect the physiological
state and growth of bacteria (Jurgens & Matz
2002).
Protozoa as indicators of activated-sludge
performance
Ciliates are present in wastewater treatment processes in terms of large numbers of individuals and
species. Even though some ciliates are carnivorous
or omnivorous, most of these microorganisms feed
upon dispersed bacteria populations. Bacterivorous
ciliates colonizing activated sludge can be subdivided into three functional groups on the basis of
their behaviour: free-swimming ciliates, which swim
in the liquor phase; crawling ciliates, which move on
the surface of the sludge floc; and sessile ciliates,
which are firmly attached to the sludge floc. Studies
of the dynamics and succession of protozoa in
activated sludge have suggested that flagellates
predominate in the system in the early stages only
because of their lower energy requirements (Curds
1966). As the flagellates decrease, they are replaced
by free-swimming ciliates and then by crawling and
attached ciliates. Three phases in the time span can
be identified from the beginning to the stabilization
of the system (Madoni 1982; Madoni & Antonietti
1984). The plant start-up phase is characterized by
the presence of species typical of raw sewage: freeswimming bacterivorous ciliates and small heterotrophic flagellates. These microorganisms cannot be
considered typical of these environments because
they are not linked to the presence of sludge flocs.
With the growth of sludge, they are replaced by
other functional groups. The second phase is
characterized by the growth of ciliates typical of the

activated-sludge habitat: crawling and attached

ciliates. In this phase, a species-rich community can
be observed, but its species structure changes with
the progressive formation of activated sludge. The
third phase (the stabilization phase) is characterized
by a ciliate community whose structure reflects the
stable condition of the aeration tank environment,
with a balance between the organic loading and the
sludge that is produced, removed and recycled. A
fully functioning plant need not host species characteristic of one of the colonization phases, unless
dysfunctions, due to the amount of sludge, the
degree of aeration, the sewage retention time, and
the organic load at the input, cause regression in the
environmental conditions (Madoni 1982). For
example, crawling ciliates (hypotrichs) decrease
with increasing organic loading (no hypotrichs are
observed in sludge loaded above 0.6 kgBOD5 kg
MLSS1 day1), while attached ciliates (peritrichs)
are able to grow over a large range of sludge loadings
(Curds & Cockburn 1970b). Of the attached
ciliates, Vorticella convallaria and V. microstoma
characterize the first phase of colonization, but the
latter is replaced by V. convallaria, which may reach
high numbers during the second and third sludge
phases. In the case of a drastic reduction in the
dissolved oxygen (DO) content of the mixed liquor,
an alternation of these two species can be observed
due to their different degrees of tolerance to the lack
of oxygen. Large numbers of V. microstoma thus
indicate a poorly aerated sludge (Madoni & Antonietti 1984). Low numbers of Opercularia often occur
in activated sludge; these attached ciliates increase
in number when the activated sludge performance
is poor due to their association with high final effluent BOD5 concentrations (Curds & Cockburn
1970b; Klimowicz 1970; Esteban et al. 1991;
Salvad et al. 1995). Testate amoebae, mostly
Arcella and Euglypha, are normally found in the
aeration tanks of activated-sludge plants that operate the biological removal of nitrogen. These plants
are characterized by low loading, a long sludge
retention time and high oxygen content (Poole
1984; Madoni et al. 1993). Under these conditions,
the quality of the final effluent is excellent and the
plant is considered to have a high biological
performance. The number of ciliates living in a
normally functioning activated-sludge plant is over
103 ml1, but the number and diversity of the ciliate
communities change according to the quality of the
settled sewage and the operating conditions of the
plant (Drakides 1980; Esteban et al. 1991; Esteban
& Tellez 1992). The most common limiting conditions are generally the presence of a shock load of
toxic material, a lack of aeration, and excess sludge

Protozoa in wastewater treatment

wastage. Since the species and functional groups of
the protozoa depend on the environmental conditions
in the aeration tank, the structure of the protozoa
community can be considered a valid indicator of
purification plant performance. Any major variations
in the plant performance are thus indicated by the
dominant group of protozoa. For this reason, the
routine analysis of these eukaryotic microorganisms
is becoming increasingly common to determine
activated-sludge plant performance. Curds and
Cockburn (1970b) were probably the first to use
protozoa as indicators of the effluent quality of
activated-sludge plants. In recent years, the biotechnology of activated-sludge processes has improved
and important innovations have been made (i.e.
tertiary treatment or advanced processes, such as
biological phosphorous and nitrogen removal).
These new processes are often incorporated in the
plant between the aeration tank and the final clarifier. The management of these processes and their
associated problems in the final clarifier (i.e. rising,
unfit sludge recycle rate) can affect the effluent quality.
Since these symptoms originate downstream from
the aeration tank (in which the microorganisms
develop), the ciliate community is unable to indicate
them. At present, protozoa are effectively used to
indicate changes in the performance of specific
activated-sludge plants (Al-Shahwani & Horan
1991; Esteban et al. 1991), but these methods cannot
always be applied directly to other, similar plants.
An objective index, based on the protozoa
community and applicable to all types of activatedsludge plants, has been proposed for the evaluation
of the biological performance of the sludge (Madoni
1994b). The method, called the Sludge Biotic
Index (SBI), is based on two principles. First, the
dominance of the microfauna key groups changes in
relation to the environmental and operational
conditions of the plant. Second, the number of
morphological species is reduced as the plant

performance deteriorates. The SBI method was set

up to investigate the relationship between the
microfauna groups and the principal physicochemical
and operational parameters. The high correlations
obtained (Table II) made it possible to select and
group the microfauna organisms into positive and
negative key groups. Positive key groups are crawling and attached bacterivorous ciliates, and testate
amoebae; negative key groups are small heterotrophic flagellates, free-swimming bacterivorous ciliates, and the peritrich ciliates Vorticella microstoma,
V. infusionum and Opercularia spp. The density and
diversity of the microfauna, moreover, were shown
to be highly correlated to the plant performance.
The advantage of this method over others is that it
provides numerical values, which enable the operator
to monitor the prevalent plant operating conditions
on a daily basis.
Effect of toxicants on protozoa in sewage
treatment processes
In recent years, the presence of toxicants in both
surface waters and sewage has become a common
occurrence. Originating mainly in industrial sewage,
they affect the performance of biological wastewater
treatment processes, reducing or inhibiting the activity
of microorganisms. Numerous papers have been
published which deal with the occurrence of heavy
metals in the various sections of sewage treatment
systems (Yetis & Gokcay 1989; Cimino & Caristi
1990; Dilek & Yetis 1992; Melcer et al. 1992;
Mazierski 1995). The effect of metal pollutants on
activated-sludge microorganisms varies with the
concentration and exposure time. The toxic effects
of copper on ciliate communities in activated-sludge
plants have been studied using concentrations from
1 to 10 mg l1 (Gracia et al. 1994). The mean total
number of ciliates and the specific diversity were
reduced by the toxic action of Cu. However,

Table II. Pearson correlation test between protozoa and plant operational conditions, obtained from 44 activated-sludge plants (from
Madoni 1994b; = negative correlation, * P < 0.01, ** P < 0.001).

Small flagellates
Free-swimming ciliates
Crawling ciliates
Attached ciliates
Vorticella microstoma
and/or V. infusionum
Opercularia spp.
Testate amoebae
Microfauna abundance
Number of species

D.O.

Nitrifying ability

BOD5 removed

Effluent colour

Sludge age

MLSS

0.652**
0.651**
0.616**
0.340

0.596**
0.549**
0.620**
-0.029

0.798**
0.829**
0.784**
0.432*

0.836**
0.725**
0.611**
0.516**

0.583**
0.668**
0.534**
0.171

0.736**
0.753**
0.534**
0.266

0.676**
0.745**
0.727**
0.626**
0.778**

0.504**
0.597**
0.912**
0.429*
0.645**

0.679**
0.763**
0.760**
0.762**
0.923**

0.648**
0.676**
0.583**
0.768**
0.841**

0.638**
0.008
0.464**
0.335
0.487**

0.722**
0.181
0.464**
0.495**
0.591**

P. Madoni

detailed observations of copper-induced reduction

of organism concentration have shown that not all
species are reduced in the same way, causing
changes in the dynamics of the ciliate communities
that inhibit the precise calculation of the median
tolerance-limit concentration. In experiments using
the free-swimming ciliate Tetrahymena pyriformis,
copper above 200 mg l1 inhibit growth significantly
(Nicolau et al. 1999). In a study in which ciliate
species were monitored in relation to metal concentration over a two-year period (Abraham et al.
1997), it was found that the major ciliate species
present were able to tolerate high concentrations of
contaminant metals, including Fe (>2 mg l1), Zn
(>0.5 mg l1), Cu (>0.06 mg l1) and Cr (0.1 mg l1).
This suggests that acclimatization can reduce the
toxicity of heavy metals upon ciliates. Moreover,
increasing the sludge age favours the retention of
heavy metals in the biofloc, thus reducing their
toxicity (Neujeed & Herman 1975). The cytotoxic
effect of the heavy metals Cd, Zn and Cu on three
different species of ciliated protozoa (Drepanomonas
revoluta, Uronema nigricans and Euplotes sp.) isolated
from an urban wastewater treatment plant has been
studied (Martin-Gonzales et al. 2006). The order of
toxicity was Cd > Cu_Zn or Cu > Cd_Zn, depending on the microbial species. In bimetallic (Cd + Zn)
treatments, the results indicated that, in general, the
presence of Zn in the same medium decreased Cd
cytotoxicity. Both cellular assays and microscopic
observations have shown that bioaccumulation is an
important mechanism of resistance to these toxic
environmental pollutants in such eukaryotic microorganisms. However, bioaccumulation might not be
the main mechanism involved in Cu resistance.
Acute toxicity tests of five heavy metals on the
protozoa community inhabiting activated sludge
have also been performed (Madoni et al. 1996).
The experimental results demonstrated the relative
toxicity of the tested metals, and indicated that the
order of toxicity of the five metals on the studied
microbial community was generally: Cd, Cu > Pb >
Zn > Cr. Lethal concentrations (24-h LC50) of
heavy metals on ciliates from activated-sludge
plants have been determined during different acute
toxicity tests (Madoni et al. 1992, 1994, 1996;
Madoni 2000; Madoni & Romeo 2006), and the
values obtained are reported in Table III. Large
differences appeared in the sensitivity of the species
to the metals. Crawling ciliates such as Chilodonella
uncinata and Trochilia minuta showed a high sensitivity to all the studied metals, while the attached
ciliates Opercularia coarctata and O. minima were
the most tolerant species. Nevertheless, the results
obtained in these studies suggest large differences in

the tolerance levels of the tested ciliated protozoan to

heavy metals. Euplotes aediculatus and Spirostomum
teres showed the highest sensitivity to nickel, while
Blepharisma americanum showed a high sensitivity to
copper. The derived toxicity revealed in these studies is of potential relevance for the microbial food
web in wastewater treatment plants since sewage
water often contains considerable amounts of toxic
metals.
Toxic substances other than the heavy metals can
also enter the plant and damage the protozoa
community. The effects of the shock load of salt
(NaCl) on protozoa communities have also been
evaluated (Salvad et al. 2001), and the results have
shown that salt concentrations from 3000 to 10,000
mg l1 gradually affected the microbial community
and few protozoa survived to 96 h. The attached
ciliates Vorticella spp. and Opercularia articulata
resisted the highest dosages of NaCl better than the
other ciliates. The toxic effect of chemical disinfection of sewage treatment plants has been tested on
three ciliates inhabiting both the receiving water
bodies and the activated sludge (Madoni et al.
1998). Three chemical disinfectants were tested:
sodium hypochlorite (NaClO), chlorine dioxide
(ClO2) and peracetic acid (PAA). The effluent
treated with ClO2 was highly toxic for Spirostomum
teres but was only slightly toxic for the other two
ciliate species. The effluent treated with NaClO had
a moderate toxic effect only on Euplotes patella. The
free-swimming ciliate Dexiostoma campylum showed
the lowest sensitivity to all the chemical disinfectants. Rehman et al. (2008) reported that the ciliates
Stylonychia mytilus and Paramecium caudatum are
resistant to the organophosphate endosulfan and
have the capacity to utilize it as a carbon source.
This allows these ciliates to be used for bioremediation of toxic xenobiotics. The introduction of
phenol to the wastewater resulted in a change in the
dominance of the sessile species, favouring the
dominance of Opercularia sp. rather than Vorticella
sp. (Papadimitriu et al. 2007). Phenol enhanced the
abundance of the suctorian Podophrya sp. and the
free-swimming Colpidium sp., the latter being typical
of wastewater treatment plants receiving industrial
inputs. The suctorian Podophrya sp. showed a
significant negative correlation to the removal of
phenol in the effluent, suggesting that its presence
may be an indicator of phenol removal efficiency.
The introduction of cyanide to the wastewater
increased the abundance of Opercularia sp. and
Colpidium sp. suggesting their tolerance under
highly toxic conditions. Chilodonella sp. showed a
negative correlation to the cyanide content of the
effluent (Papadimitriu et al. 2007).

Protozoa in wastewater treatment

Table III. 24-h LC50 mean values (mg l1) of some protozoa from activated sludges tested with heavy metals.
Species
Aspidisca cicada
A. cicada
A. cicada
A. cicada
A. cicada
A. cicada
A. lynceus
A. lynceus
A. lynceus
A. lynceus
A. lynceus
Blepharisma americanum
B. americanum
B. americanum
B. americanum
Chilodonella uncinata
C. uncinata
C. uncinata
C. uncinata
C. uncinata
Colpidium colpoda
C. colpoda
C. colpoda
C. colpoda
C. colpoda
Dexiostoma campylum
D. campylum
D. campylum
D. campylum
D. campylum
D. campylum
Drepanomonas revoluta
D. revoluta
Euplotes affinis
E. affinis
E. affinis
E. affinis
E. affinis
E. affinis
E. patella
E. patella
E. patella
E. patella
E. patella

Metal

LC50

Cd
Cu
Hg
Zn
Pb
Cr
Cd
Cu
Cr
Pb
Zn
Cd
Cu
Hg
Zn
Cd
Cu
Cr
Pb
Zn
Cd
Cu
Cr
Ni
Pb
Cd
Cu
Cr
Hg
Ni
Pb
Cr
Pb
Cd
Cu
Cr
Hg
Pb
Zn
Cd
Cu
Cr
Hg
Ni

0.31
0.021
0.07
2.4
1.26
2.35
2.21
1.82
14.5
0.004
0.05
1.4
0.001
0.017
1.05
<0.0001
<0.00004
0.011
<0.0015
<0.0002
0.89
0.05
108
1.19
0.23
0.2
0.01
3.29
0.017
1.05
1.1
45.6
0.88
0.4
0.064
2.73
0.19
2.32
3.10
2.65
0.011
9.47
0.13
7.70

Ref.
a
a
a
a
b
b
c
c
c
c
c
a
a
a
a
c
c
c
c
c
d
d
d
d
d
a
a
b
a
e
b
b
b
a
a
b
a
b
a
a
a
b
a
e

Species

Metal

LC50

Ref.

E. patella
E. aediculatus
E. aediculatus
E. aediculatus
E. aediculatus
E. aediculatus
Opercularia coarctata
O. coarctata
O. coarctata
O. coarctata
O. minima
O. minima
O. minima
O. minima
Paramecium caudatum
P. caudatum
P. caudatum
P. caudatum
P. caudatum
P. caudatum
P. caudatum
Spirostomum teres
S. teres
S. teres
Trochilia minuta
T. minuta
T. minuta
T. minuta
Uronema nigricans
U. nigricans
U. nigricans
U. nigricans
U. nigricans
U. nigricans
Vorticella convallaria
V. convallaria
V. convallaria
V. convallaria
V. octava
V. octava
V. octava
V. octava
V. octava

Pb
Cd
Cu
Cr
Ni
Pb
Cd
Cr
Pb
Zn
Cd
Cr
Pb
Zn
Cd
Cu
Cr
Hg
Ni
Pb
Zn
Cr
Ni
Pb
Cd
Cu
Cr
Zn
Cd
Cu
Cr
Hg
Pb
Zn
Cd
Cr
Pb
Zn
Cd
Cr
Cu
Pb
Zn

2.18
0.59
0.01
0.10
0.03
0.50
3.75
211
3.28
10.3
5.55
164
5.0
84.7
0.18
0.011
2.57
0.02
0.49
2.26
2.50
3.23
0.17
1.08
0.27
0.31
9.25
0.20
0.62
0.014
2.18
0.004
1.62
0.003
3.81
101
2.29
3.79
3.24
80
2.05
2.81
0.57

b
d
d
d
d
d
c
c
c
c
c
c
c
c
a
a
b
a
e
b
a
b
e
e
c
c
c
c
a
a
b
a
b
a
c
c
c
c
c
c
c
c
c

The letters in the bibliography columns refer to published references, as follows: a, Madoni et al. (1992); b, Madoni et al. (1994); c,
Madoni et al. (1996); d, Madoni & Romeo (2006); e, Madoni (2000).

These studies of ciliate sensitivity to a wide number

of toxic substances provide a yardstick for identifying
the intensity and potential for ecological damage
caused by anthropogenic pollutants discharged into
surface waters.
References
Abraham JV, Butler RD, Sigee DC. 1997. Ciliate populations
and metals in an activated-sludge plant. Water Research
31:11031111.

Agersborg HPK, Hatfield WD. 1929. The biology of a sewagetreatment plant A preliminary survey. Sewage Works Journal
1:411415.
Al-Shahwani SM, Horan NJ. 1991. The use of protozoa to indicate changes in the performance of activated sludge plants.
Water Research 25:633638.
Amann R, Lemmer H, Wagner M. 1998. Monitoring the
community structure of wastewater treatment plants:
A comparison of old and new techniques. FEMS Microbial
Ecology 25:205215.
Antonietti R, Madoni P, Ghetti PF. 1981. Some notes on the biological self-purification process: Biological water-treatment

10

P. Madoni

plants as man-made ecosystems. Societ Italiana di Ecologia,

Atti 1:379382.
Ardern E, Lockett WT. 1936. Laboratory tests for ascertaining the condition of activated sludge. Water Pollution Control 35:212215.
Augustin H, Foissner W. 1992. Morphologie und kologie einiger Ciliaten (Protozoa: Ciliophora) aus dem Belebtschlamm.
Archiv fr Protistenkunde 141:243283.
Barker AN. 1943. The protozoan fauna of sewage disposal plants.
Naturalist, Hull 806:6569.
Bloem J, Ellenbroek F, Br-Gilissen MJB, Cappenberg TE. 1989.
Protozoan grazing and bacterial production in stratified Lake
Vechten estimated with fluorescently labeled bacteria and by
thymidine incorporation. Applied and Environmental Microbiology 55:17871795.
Bloem J, Starink M, Br-Gilissen MJB, Cappenberg TE. 1988.
Protozoan grazing, bacterial activity, and mineralization in
two-stage continuous cultures. Applied and Environmental
Microbiology 54:31133121.
Brown TJ. 1965. A study of the protozoa in a diffused-air
activated-sludge plant. Water Pollution Control 64:375378.
Chen S, Xu M, Cao H, Zhu J, Zhou K, Xu J, Yang X, Gan Y,
Liu W, Zhai J, Shao Y. 2004. The activated-sludge fauna and
performance of five sewage treatment plants in Beijing, China.
European Journal of Protistology 40:147152.
Chung J, Strom P. 1991. Microbiological study of ten New Jersey
rotating biological contactor wastewater treatment plants.
Research Journal of the Water Pollution Control Federation
63:3543.
Cimino G, Caristi C. 1990. Acute toxicity of heavy metals to
aerobic digestion of waste cheese whey. Biological Wastes
33:201210.
Clay E. 1964. The fauna and flora of sewage processes. 2. Species
list. I.C.I. Ltd, Paints Division, Research Dept Memorandum
PVM 45/A/732.
Coleman DC, Cole CV, Hunt HV, Klein DA. 1978. Trophic interactions in soils as they affect energy and nutrient dynamics: Part
I, introduction. Microbial Ecology 4:345349.
Curds CR. 1966. An ecological study of the ciliated protozoa in
activated-sludge process. Oikos 15:282289.
Curds CR. 1975. Protozoa. In: Curds CR, Hawkes HA, editors.
Ecological aspects of used-water treatment. London:
Academic Press. pp. 203268.
Curds CR. 1982. The ecology and role of protozoa in aerobic
sewage treatment processes. Annual Review of Microbiology
36:2746.
Curds CR, Cockburn A. 1970a. Protozoa in biological sewagetreatment processes I. A survey of the protozoan fauna of
British percolating filters and activated sludge of sewage and
waste treatment plants. Water Research 4:225236.
Curds CR, Cockburn A. 1970b. Protozoa in biological sewagetreatment processes II. Protozoa as indicators in the
activated sludge process. Water Research 4:237249.
Curds CR, Cockburn A, Vandyke JM. 1968. An experimental
study of the role of the ciliated protozoa in the activated
sludge process. Water Pollution Control 67:312329.
Curds CR, Fey GJ. 1969. The effect of ciliated protozoa on fate
of Escherichia coli in the activated sludge process. Water
Research 3:853867.
Dilek FB, Yetis U. 1992. Effects of heavy metals on activatedsludge process. Water Science and Technology 26:801813.
Drakides C. 1980. La microfaune des boues activs. Etude dune
mthode dobservation et application au suivi dun pilote en
phase de dmarrage. Water Research 14:11991207.
Esteban C, Tellez C. 1992. The influence of detergents on the
development of ciliate communities in activated sludge.
Water, Air and Soil Pollution 61:185190.

Esteban G, Tellez C, Bautista LM. 1991. Effect of habitat quality

on ciliated protozoa communities in sewage treatment plants.
Water Research 25:967972.
Ettl M. 2001. The ciliate community (Protozoa: Ciliophora) of a
municipal activated sludge plant: Interactions between species
and environmental factors. Protozoological Monographs 1:162.
Fairbrother TH, Renshaw A. 1922. The relation between chemical
constitution and antiseptic action in the coal tar dyestuffs. Journal of the Society of Chemical Industry, London 41:134138.
Foissner W, Berger H. 1996. A user-friendly guide to the ciliates
(Protozoa, Ciliophora) commonly used by hydrobiologists as
bioindicators in rivers, lakes, and waste waters, with notes on
their ecology. Freshwater Biology 35:375482.
Gracia MP, Salvado H, Rius M, Amigo JM. 1994. Effects of copper on ciliate communities from activated sludge plants. Acta
Protozoologica 33:219226.
Horan NJ. 1990. Biological wastewater treatment systems. Theory and operation. Chichester: John Wiley and Sons.
Hul M. 1992. The composition of microfauna of the biofilm of a
rotating biological disc and rotating biological multicage
under conditions of high loading with pollutants. Acta Hydrobiologica 34:129138.
Jurgens K, Matz C. 2002. Predation as a shaping force for the
phenotypic and genotypic composition of planktonic bacteria.
Antoine Van Leeuwenhoek 81:413434.
Kinner NE, Curds CR. 1987. Development of protozoan and
metazoan communities in rotating biological contactor biofilms. Water Research 21:481490.
Klimowicz H. 1970. Microfauna of activated sludge. Part I:
Assemblage of microfauna in laboratory models of activated
sludge. Acta Hydrobiologica 12:357376.
Luna-Pabello VM, Aladro-Lubel A, Duran-de-Bazua C. 1992.
Temperature effect on ciliates diversity and abundance in a
rotating biological reactor. Bioresource Technology 39:5560.
Madoni P. 1981. I Protozoi Ciliati degli Impianti Biologici di
Depurazione. Rome: CNR AQ/1/167.
Madoni P. 1982. Growth and succession of ciliate populations
during the establishment of a mature activated sludge. Acta
Hydrobiologica 24:223232.
Madoni P. 1994a. Quantitative importance of ciliated protozoa in
activated sludge and biofilm. Bioresource Technology
48:245249.
Madoni P. 1994b. A sludge biotic index (SBI) for the evaluation
of the biological performance of activated sludge plants based
on the microfauna analysis. Water Research 28:6775.
Madoni P. 2000. The acute toxicity of nickel to freshwater ciliates. Environmental Pollution 109:5359.
Madoni P. 2002. Protozoa in activated sludge. In: Bitton G, editor. Encyclopedia of environmental microbiology. New York:
John Wiley & Sons. pp. 26052612.
Madoni P. 2003. Protozoa as indicators of wastewater treatment
efficiency. In: Mara D, Horan N, editors. The handbook of
water and wastewater microbiology. London: Academic
Press. pp. 361371.
Madoni P, Antonietti R. 1984. Colonization dynamics of ciliated
protozoa populations in an activated sludge plant. Proceeding
IV Italian Symposium of Population Dynamics, Parma, 24
October 1981. Parma: Italian Society of Population Dynamics. pp. 105112.
Madoni P, Davoli D, Chierici E. 1993. Comparative analysis of
the activated sludge microfauna in several sewage treatment
works. Water Research 27:14851491.
Madoni P, Davoli D, Gorbi G. 1994. Acute toxicity of lead, chromium, and other heavy metals to ciliates from activated sludge
plants. Bulletin of Environmental Contamination and Toxicology 53:420425.

Protozoa in wastewater treatment

Madoni P, Davoli D, Gorbi G, Vescovi L. 1996. Toxic effect of
heavy metals on the activated sludge protozoan community.
Water Research 30:135141.
Madoni P, Esteban G, Gorbi G. 1992. Acute toxicity of cadmium, copper, mercury, and zinc to ciliates from activated
sludge plants. Bulletin of Environmental Contamination and
Toxicology 49:900905.
Madoni P, Ghetti PF. 1981. The structure of ciliated protozoa
communities in biological sewage treatment plants. Hydrobiologia 83:207215.
Madoni P, Ghetti PF, Antonietti R. 1979. Analisi delle caratteristiche biologiche di un impianto pilota a tamburo rotante.
Inquinamento 21:4952.
Madoni P, Gorbi G, Taj E. 1998. Toxic effect of chemical
disinfection of wastewater on freshwater ciliates. Acta Protozoologica 37:221225.
Madoni P, Romeo MG. 2006. Acute toxicity of heavy metals
towards freshwater ciliated protists. Environmental Pollution
141:17.
Mallory LM, Yuk CS, Liang LN, Alexander M. 1983. Alternative prey: A mechanism for elimination of bacterial species
by protozoa. Applied and Environmental Microbiology
46:10731079.
Martin-Cereceda M, Prez-Uz B, Serrano S, Guinea A. 2002. An
integrate approach to analyse biofilms of a full scale wastewater treatment plant. Water Science and Technology 46:199
206.
Martin-Cereceda M, Serrano S, Guinea A. 1996. A comparative
study of ciliated protozoa communities in activated-sludge
plants. FEMS Microbiology Ecology 21:267276.
Martin-Gonzales A, Diaz S, Borniquel S, Gallego A, Gutierrez
JC. 2006. Cytotoxicity and bioaccumulation of heavy metals
by ciliated protozoa isolated from urban wastewater treatment
plants. Research in Microbiology 157:108118.
Mazierski J. 1995. Effect of chromium (CrVI) on the growth rate
of activated sludge bacteria. Water Research 29:14791482.
Melcer H, Monteith H, Nutt SG. 1992, Activated sludge process
response to variable inputs of heavy metals. Water Science
and Technology 25:387394.
Mistri M, Gavinelli M, Gorni A. 1994. Structure of a protozoan
community in a percolating filter. Bollettino di Zoologia
61:6571.
Morisita I. 1970. Studies on protozoa populations in activated
sludge of sewage and waste treatment plants. Japanese Journal
of Protozoology 3:16.
Neujeed RD, Herman ER. 1975. Heavy metal removal by
acclimated activated sludge. Journal of the Water Pollution
Control Federation 47:310329.

11

Nicolau A, Mota M, Lima N. 1999. Physiological responses of

Tetrahymena pyriformis to copper, zinc, cycloheximide and
Triton X-100. FEMS Microbiology Ecology 30:209216.
Nisbet B. 1984. Nutrition and feeding strategies in protozoa.
London: Croom Helm.
Papadimitriou C, Palaska G, Lazaridou M, Samaras P, Sakellaropoulos GP. 2007. The effects of toxic substances on the activated sludge microfauna. Desalination 211:177191.
Prez-Uz B, Franco C, Martin-Cereceda M, Arregui L, Campos
I, Serrano S, et al. 1998. Biofilm characterization of several
wastewater treatment plants with rotating biological contactors in Madrid (Spain). Water Science and Technology
37:215218.
Petropoulos P, Gilbride KA. 2005. Nitrification in activated sludge
batch reactors is linked to protozoan grazing of the bacterial
population. Canadian Journal of Microbiology 51:791799.
Pogue AJ, Gilbride KA. 2007. Impact of protozoan grazing on
nitrification and the ammonia- and nitrite-oxidizing bacterial
communities in activated sludge. Canadian Journal of Microbiology 53:559571.
Poole JEP. 1984. A study of the relationship between the mixed
liquor fauna and plant performance for a variety of effluent
quality in activated sludge sewage treatment works. Water
Research 18:281287.
Ratsak CH, Maarsen KA, Kooijman SAL. 1996. Effects of protozoan on carbon mineralization in activated sludge. Water
Research 30:112.
Rehman A, Shuja R, Shakoori AR. 2008. Tolerance of two
ciliates, Stylonychia mytilus and Paramecium caudatum, isolated from industrial effluents to an organophosphate,
endosulfan and their potential use in bioremediation of
insecticide-contaminated wastewater. Pakistan Journal of
Zoology 40:255261.
Salvad H, Gracia MP, Amig JM. 1995. Capability of ciliated
protozoa as indicators of effluent quality in activated sludge
plants. Water Research 29:10411050.
Salvad H, Mas M, Menndez S, Gracia MP. 2001. Effects of
shock loads of salt on protozoan communities of activated
sludge. Acta Protozoologica 40:177185.
Tezuka Y. 1990. Bacterial regeneration of ammonium and
phosphorus ratio of organic substrates. Microbial Ecology
29:227238.
Verhagen FJM, Laanbroek HG. 1991. Competition for ammonium between nitrifying and heterotrophic bacteria in dual
energy-limited chemostats. Applied and Environmental
Microbiology 57:32553263.
Yetis U, Gokcay CF. 1989. Effect of nickel (II) on activated
sludge. Water Science and Technology 23:10031007.